Sample preparation for nucleic acids

Abstract

Sample preparation compositions and methods for purifying plasmid DNA from biological samples is provided, are provided. The compositions and methods provided herein allow pDNA analysis to be carried out without centrifugation. The preparation process is amenable to high throughput processing using manual or robotic platforms.

Description

Attorney Docket No. TP387448WO1 SAMPLE PREPARATION FOR NUCLEIC ACIDS FIELD

[0001] The present teachings generally relate to compositions, processes, methods, and kits for preparation of nucleic acids, and in particular, for the preparation of nucleic acids propagated in vivo, e.g., in gram negative microorganisms. INTRODUCTION

[0002] Production of nucleic acids such as plasmid DNA are often scaled by propagation in vivo. For example, the gram-negative bacterium Escherichia coli is commonly used as a host to propagate plasmid DNA (pDNA), which is then used in downstream applications, e.g., gene therapy. Endotoxin, or Lipopolysaccharide (“LPS”) is the major constituent of the outer membrane of Gram-negative bacteria. LPS is known to trigger inflammatory responses in mammals and, thus, represents the endotoxic principle of LPS. Nucleic acid preparations from E. coli are generally contaminated with endotoxin. Removal of bacterial endotoxin from nucleic acid preparations thus can be a challenging and expensive process that has been necessary to ensure the safety of the final product, e.g., therapeutic nucleic acids. Furthermore, it is desirable to automate processes and to provide eco-friendly solutions to the extent possible when scaling production. It is thus desirable to have a process for producing pDNA that advantageously remove endotoxin and that are readily automated. Accordingly provided herein are compositions and method of purification of plasmid DNA that do not involve, e.g.., centrifugation, and that minimize or eliminate the use of non-REACH compliant agents. SUMMARY

[0003] Provided herein are methods for preparing nucleic acids such as plasmid DNA (pDNA) from samples, and kits and compositions for such methods. The embodiments described herein are based, in part, upon the surprising discovery of buffers and systems for nucleic acid (e.g. pDNA) preparations that are eco-friendly (e.g., are REACH compliant), produce pDNA suitable for therapeutic applications (e.g., are endotoxin-free), and at the sameAttorney Docket No. TP387448WO1 time are readily amenable to automation (e.g., can be used in workflows that eliminate the need for centrifugation). Accordingly, in one aspect, provided herein are compositions useful in the preparation of nucleic acids such as pDNA comprising, in aqueous solution, a non-ionic detergent in amounts of about 0.1% to about 25%; and a polymyxin at a concentration of about 0.5 mg / mL to about 15 mg / mL. The compositions can further include an alkali or alkaline earth metal salt at concentrations of about 0.1 M to about 1.5 M and a buffering agent at a concentration between about 10 mM to about 100 mM.

[0004] Exemplary non-ionic detergents useful in the compositions and methods provided herein include Ecosurf™EH-9, Ecosurf™SA-4, Ecosurf™SA-9, Ecosurf™EH-6, Ecosurf™EH-3, Saponin, Ecosurf™, Tween®80, Tween®85, Tween®40, Tween®20, Tween®60, Tween®65, Tergitol 15-S-7, Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15- S-40, Tergitol NP-11, Tergitol NP-13, Tergitol NP-50, Tergitol NP-30, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S-20, Tergitol NP-70, Tergitol NP- 40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S-5, Pluronic F-127, Synperonic®F 108, Synperonic®PE P105, SA-7, Poloxamer 188, , Triton X-45, Triton X- 100, Triton X-114, Triton X-102, Triton X-165, Triton X-305, Triton X-705, Triton™X-405, Triton™X-405, reduced, Triton™X-100 reduced, Triton™N-101, reduced, Triton™CG-110, Brij®35, Brij®58, Brij®L23, Brij®S10, BRIJ®O20, Brij®S 100, Brij®O10, Brij®S20, Brij®C10, Brij®L4, Brij®93, SP Brij®S2 MBAL, Digitonin, MERPOL®A, MERPOL®HCS, MERPOL®SH, MERPOL®SE, Elugent, Octyl β-D-glucopyranoside, n-Dodecyl β-D- maltoside, Decyl β-D-maltopyranoside, n-Octyl β-D-maltoside, Decyl β-D-glucopyranoside, Octyl α-D-glucopyranoside, Hexyl β-D-glucopyranoside, Nonyl β-D-maltoside, IGEPAL®CA-630, IGEPAL®CO-520, IGEPAL®CO-630, IGEPAL®CA-720, IGEPAL®CO-890, Octyl-beta-Glucoside, Octylthio Glucoside, cocoamide monoethanolamine (Cocamide MEA), cocamide diethanolamine (Cocamide DEA), or any combination thereof. Preferably, the non-ionic detergent is REACH compliant.

[0005] Polymyxins useful in the compositions and methods provided herein include, e.g., polymyxin-B and polymyxin-E.Attorney Docket No. TP387448WO1

[0006] Exemplary alkali or alkaline earth metal salt useful in the compositions and methods provided herein include LiCl, NaCl, KCl, BaCl2, MgCl2, and CaCl2, and corresponding acetates, or any combination thereof.

[0007] Buffering agents useful in the compositions and methods provided herein include N- 2-acetamido-2-aminoethanesulfonic acid (ACES), N-2-acetamido-2-iminodiacetic acid (ADA), 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid (AMPSO), N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES), 4-cyclohexylamino-1-butane sulfonic acid (CABS), 3-cyclohexylamino-1-propane sulfonic acid (CAPS), 3- cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO), 2-N- cyclohexylaminoethanesulfonic acid (CHES), 3-N,N-bis-2-hydroxyethylamino-2- hydroxypropanesulfonic acid (DIPSO), N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS or HEPPS), N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS), 4-N- morpholinobutanesulfonic acid (MOBS), 3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO), Bis-Tris, hydroxyethylpiperazine ethane sulfonic acid (HEPES), morpholinoethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), [tris(hydroxymethyl)methylamino] propanesulfonic acid (TAPS), N-trishydroxymethyl- methyl-4-aminobutanesulfonic acid (TABS), N-trishydroxymethyl-methyl-3- aminopropanesulfonic acid (TAPS), 3-N-trishydroxymethyl-methylamino-2- hydroxypropanesulfonic acid (TAPSO), N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES), N-(2-acetamido)iminodiacetic acid (ADA), Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), Tris-Hydrochloride (Tris-HCl), N-trishydroxymethylmethylglycine (TRICINE), or any combination thereof.

[0008] The compositions provided and provided herein can be provided alone or with additional compositions or buffers. For example, the compositions provided herein can be provided including for example, one or more of: a lysis buffer, which includes a denaturing agent; a re-suspension buffer including a RNase; a precipitation buffer, which includes an alkali or alkaline metal salt (e.g., an acetate); one or more wash buffers; one or more solid phases, and an elution buffer.Attorney Docket No. TP387448WO1

[0009] In another aspect, provided herein are methods for preparing nucleic acids (e.g., pDNA) from a biological sample (e.g., a culture of E. coli or other gram-negative bacteria, or respiratory gram-positive bacteria, harboring pDNA). Accordingly, provided herein are methods for preparing pDNA from a sample containing microorganisms. The method can include contacting a sample including microorganism(s) containing pDNA with a first solid phase under conditions whereby the beads bind the microorganisms. The microorganism- bound first solid phase can be removed from the remainder of the sample and resuspended in a resuspension buffer comprising RNase. A lysis buffer can be added to the microorganism- bound first solid phase to produce a lysate. The lysis buffer and the resuspension buffer can be added at the same time, or sequentially. For example, the resuspension buffer comprising the RNase can be added prior to the lysis buffer, or the resuspension buffer comprising the RNase can be added after the lysis buffer. The lysis buffer can contain a strong base or other denaturing agent to facilitate lysis of the microorganism and to release the nucleic acids, e.g., pDNA. The microorganism-bound solid phase can be incubated in the resuspension / lysis buffer mixture at a temperature, e.g., from about 5 °C to about 40 °C, from about 15 °C to about 30 °C, from about 16 °C to about 28 °C or from about 19 °C to about 25 °C as further described infra for a period of time, e.g., between at least 1 minute to one hour or longer (for example, up to 24 hours).

[0010] A precipitation buffer can be added to the lysate. The precipitation buffer can contain an alkali or alkaline metal salt (e.g., KOAc, NaOAc, LiOAc, or the like) at concentrations of about 0.1 M to about 1.5 M (after being added to the lysate);. When mixed with the lysate, the precipitation buffer produces a first mixture comprising a precipitate comprising cellular debris. Desirably, the precipitation buffer is mixed with the lysate, e.g., by a stirring or mixing means (such as for example, movement of a rod within a well containing the mixture). The resulting mixture can be incubated at about 16 °C to about 28 °C for at least one minute.

[0011] The first solid phase can be separated from the first mixture, leaving a clarified solution. An endotoxin removal buffer (ERB) can be added to the clarified solution to produce a second mixture. The endotoxin removal buffer can contain a polymyxin and a non-ionic detergent. For example, the endotoxin removal buffer can contain a polymyxin at a final concentration of about 0.5 mg / mL to about 15 mg / mL (in the second mixture) and a non-ionicAttorney Docket No. TP387448WO1 detergent at a final concentration of about 0.1% to about 25% (in the second mixture). Optionally, the endotoxin removal buffer can have a pH of about 4.5 to about 6.5 and include a buffering agent. For example, the buffering agent can be at a final concentration of about 10 mM to about 100 mM (in the second mixture). Desirably, the endotoxin removal buffer is mixed with the clarified solution, e.g., by a stirring or mixing means (such as for example, movement of a rod within a well containing the mixture). The clarified solution and ERB can be allowed to incubate at a temperature , e.g., from about 5 °C to about 40 °C, from about 15 °C to about 30 °C, from about 16 °C to about 28 °C or from about 19 °C to about 25 °C as further described infra for a period of time, e.g., between at least 1 minute to one hour or longer (for example, up to 24 hours).

[0012] The nucleic acid (e.g., pDNA) can be recovered from the second mixture.

[0013] Recovery of the nucleic acids can advantageously be achieved by providing a second solid phase in the second mixture, to which the nucleic acids reversibly bind in the second mixture. The second mixture including the solid phase can be incubated at a temperature, e.g., from about 5 °C to about 40 °C, from about 15 °C to about 30 °C, from about 16 °C to about 28 °C or from about 19 °C to about 25 °C as further described infra for a period of time, e.g., between at least 1 minute to one hour or longer (for example, up to 24 hours).

[0014] The nucleic acid bound solid phase can be removed from the second mixture and contacted with an elution buffer, which releases the bound nucleic acid (e.g., pDNA) from the solid phase into the elution buffer. The nucleic acid bound solid phase can be incubated in the elution buffer at 16 °C to about 28 °C for at least one minute, or longer. The second solid phase can be contacted with an elution buffer at a temperature, e.g., from about 5 °C to about 40 °C, from about 15 °C to about 30 °C, from about 16 °C to about 28 °C or from about 19 °C to about 25 °C as further described infra for a period of time, e.g., between at least 1 minute to one hour or longer (for example, up to 24 hours). Preferably, the methods can include, prior to contacting the nucleic acid-bound solid phase with an elution buffer, contacting the solid phase with one or more wash buffers.Attorney Docket No. TP387448WO1

[0015] In another aspect, provided herein is a method for preparing plasmid DNA (pDNA) from a biological sample comprising a microorganism, wherein the microorganism comprises the pDNA, comprising: (a) contacting the biological sample with a first solid phase under conditions which selectively bind the microorganism; (b) incubating the microorganism- bound first solid phase with a lysis / resuspension buffer and an RNase at an incubation temperature and for a time to produce a lysate; (c) incubating the lysate with a precipitation buffer at an incubation temperature and for a time to produce a first mixture comprising cellular debris and pDNA, wherein the cellular debris selectively binds to the first solid phase; (d) removing the cellular debris-bound first solid phase from the second mixture to produce a pDNA-enriched solution; (e) adding a second solid phase and an endotoxin removal buffer to the pDNA-enriched solution to produce a second mixture, wherein the pDNA selectively binds to the second solid phase in the second mixture; and (f) eluting the pDNA from the pDNA bound second solid phase.

[0016] In another aspect, provided herein is a method for preparing plasmid DNA (pDNA) from a biological sample comprising a microorganism, wherein after step (a), the microorganism-bound first solid phase is removed from the biological sample and resuspended in the lysis buffer of step (b).

[0017] In another aspect, provided herein is a method for preparing plasmid DNA (pDNA) from a biological sample comprising a microorganism, method further comprising: (g) washing the pDNA bound second solid phase with ammonium salts; (h) washing the pDNA bound second solid phase with water; (i) contacting the second solid phase from step (d) with an elution buffer for a time and at a temperature releasing the pDNA from the second solid phase into the elution buffer; and wherein the pDNA from the endotoxin free lysate is compatible with in situ polymerase or reverse transcription reactions.

[0018] In another aspect, provided herein is a method for preparing plasmid DNA (pDNA) from a biological sample comprising a microorganism, wherein the pDNA binds the second solid phase at a pH of less than about 6.0 and is released from the second solid phase at a pH of greater than about 8.0.

[0019] In another aspect, provided herein is a method for preparing plasmid DNA (pDNA) from a biological sample comprising a microorganism, wherein the pH of the lysis buffer is between about 11.8 and about 14.0.Attorney Docket No. TP387448WO1

[0020] In another aspect, provided herein is a method for preparing plasmid DNA (pDNA) from a biological sample comprising a microorganism, wherein the pH of the precipitation buffer is between about 3.5 and about 5.5.

[0021] In another aspect, provided herein is a method for preparing plasmid DNA (pDNA) from a biological sample comprising a microorganism, wherein the pH of the elution buffer is between about 8.0 and about 9.5.

[0022] Solid phases useful in the embodiments provided herein include beads (e.g., magnetic beads, paramagnetic beads, superparamagnetic beads, polystyrene beads, ceramic beads, or the like), particles, fibers, membranes, glass, paper, slides, or the like. Desirably, the solid phase can be functionalized, e.g., with amine groups, carboxylic acid groups, or the like.

[0023] Accordingly, provided herein are kits or systems comprising at least one solid phase that comprises magnetic beads, paramagnetic beads, or superparamagnetic beads, and the use of such solid phase in the methods provided herein. The at least one solid phase can include plurality of ionizable groups selected from the group consisting of polyhydroxylated amines, histidine, polyamines, imidazole, and poly-histidine. More specifically, the at least one solid phase can include, e.g., on its surface, a plurality of ionizable groups selected from the group consisting of N-trishydroxymethylmethylglycine (TRICINE), trishydroxymethylaminomethane (Tris), polyhydroxylated imidazoles, bis-2- hydroxyethyliminotrishydroxymethylmethane (Bis-Tris), 1,3- bistrishydroxymethylmethylaminopropane (Bis-TrisPropane), N- trishydroxymethylmethylglycine (TRICINE), trishydroxymethylaminomethane (Tris), polyhydroxylated imidazoles, triethanolamine dimers and polymers, heterocyclic nitrogen- containing aromatic or aliphatic compounds and may be monomers, oligomers or polymers, such as morpholine-, pyrrole-, pyrrolidine-, pyridine-, pyridinol-, pyridone-, pyrroline-, pyrazole-, pyridazine-, pyrazine-, piperidone-, piperidine-, or piperazine-containing compounds.

[0024] In one embodiment, teachings herein include a kit comprising an endotoxin removal buffer (ERB) useful in the isolation of nucleic acids (e.g., pDNA) from a biological sample, wherein the said ERB comprises a non-ionic detergent in amounts of about 0.1% to about 25% and a polymyxin at a concentration of about 0.5 mg / mL to about 15 mg / mL. The ERBAttorney Docket No. TP387448WO1 can also include an alkali or alkaline earth metal salt at a concentration of about 0.1 M to about 1.5 M, a buffering agent at a concentration of about 10 mM to about100 mM, or both. In another embodiment, the kit can further comprise one or more of a lysis buffer, a resuspension buffer, a precipitation buffer, an elution buffer, one or more wash buffers, or any combination thereof. The lysis buffer can comprise a denaturing agent; the resuspension buffer can comprise an RNase; the precipitation buffer can comprise an alkali metal salt (such as an acetate); and the elution buffer can comprise a buffering agent and an alkali metal hydroxide. The kit can advantageously include at least one solid phase, e.g., magnetic, paramagnetic, or superparamagnetic particles or beads.

[0025] In some embodiments, the teachings here also include a composition for endotoxin removal comprising, in an aqueous solution an alkali or alkaline earth metal salt at concentrations of about 0.1 M to about 1.5 M; a non-ionic detergent in amounts of about 0.1% to about 25%; a polymyxin at concentrations of about 0.5 mg / mL to about 15 mg / mL; and a buffering agent at a concentration of about 10 mM to about100 mM.

[0026] In some embodiments, the teachings here also include a composition for endotoxin removal comprising, in an aqueous solution an alkali or alkaline earth metal salt at concentrations of about 0.1 M to about 1.5 M, wherein the alkali metal salt is selected from the group consisting of LiCl, NaCl, and KCl, or any combination thereof; a non-ionic detergent in amounts of about 0.1% to about 25%, wherein the non-ionic detergent is selected from the group consisting of Ecosurf™ EH-9, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™, Tween® 80, Tween® 85, Tween® 40, Tween® 20, Tween® 60, Tween® 65, Tergitol 15-S-7, Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15- S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, Tergitol NP-50, Tergitol NP-30, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S-20, Tergitol NP- 70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S-5, Pluronic F-127, Synperonic® F 108, Synperonic® PE P105, SA-7, Poloxamer 188, , Triton X-45, Triton X-100, Triton X-114, Triton X-102, Triton X-165, Triton X-305, Triton X-705, Triton™ X-405, Triton™ X-405, reduced, Triton™ X-100 reduced, Triton™ N-101, reduced, Triton™ CG-110, Brij® 35, Brij® 58, Brij® L23, Brij® S10, BRIJ® O20, Brij® S 100, Brij® O10, Brij® S20, Brij® C10, Brij® L4, Brij® 93, SP Brij® S2 MBAL, Digitonin, MERPOL® A, MERPOL® HCS, MERPOL® SH, MERPOL® SE, Elugent, Octyl β-D-glucopyranoside,Attorney Docket No. TP387448WO1 n-Dodecyl β-D-maltoside, Decyl β-D-maltopyranoside, n-Octyl β-D-maltoside, Decyl β-D- glucopyranoside, Octyl α-D-glucopyranoside, Hexyl β-D-glucopyranoside, Nonyl β-D- maltoside, IGEPAL® CA-630, IGEPAL® CO-520, IGEPAL® CO-630, IGEPAL® CA-720, IGEPAL® CO-890, Octyl-beta-Glucoside, Octylthio Glucoside, cocoamide monoethanolamine (Cocamide MEA), cocamide diethanolamine (Cocamide DEA), or any combination thereof; a polymyxin at concentrations of about 0.5 mg / mL to about 15 mg / mL, wherein the polymyxin is selected from the group consisting of polymyxin-B and polymyxin- E; and a buffering agent at a concentration of about 10 mM to about 100 mM, wherein the buffering agent is selected from the group consisting of N-2-acetamido-2- aminoethanesulfonic acid (ACES), N-2-acetamido-2-iminodiacetic acid (ADA), 3-1,1- dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid (AMPSO), N,N-bis2- hydroxyethyl-2-aminoethanesulfonic acid (BES), 4-cyclohexylamino-1-butane sulfonic acid (CABS), 3-cyclohexylamino-1-propane sulfonic acid (CAPS), 3-cyclohexylamino-2- hydroxy-1-propane sulfonic acid (CAPSO), 2-N-cyclohexylaminoethanesulfonic acid (CHES), 3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO), N-2- hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS or HEPPS), N-2- hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS), 4-N-morpholinobutanesulfonic acid (MOBS), 3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO), bis(2- hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris), hydroxyethylpiperazine ethane sulfonic acid (HEPES), morpholinoethanesulfonic acid (MES), 3-(N- morpholino)propanesulfonic acid (MOPS), [tris(hydroxymethyl)methylamino] propanesulfonic acid (TAPS), N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS), N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS), 3-N- trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO), N- trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES), N-(2-acetamido)iminodiacetic acid (ADA), Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), N- trishydroxymethylmethylglycine (TRICINE), or any combination thereof.

[0027] Sample preparation methods provided herein are useful for preparing nucleic acids for downstream methods wherein pDNA used in production of therapeutics, e.g., in cell and gene therapy applications.Attorney Docket No. TP387448WO1 DRAWINGS

[0028] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0029] FIG.1 provides data demonstrating that a variety of non-ionic surfactants are effective at isolating pDNA from Escherichia coli.

[0030] FIG. 2 provides ratio of absorbance at 260 nm and 280 nm (A260 / 280) and ratio of absorbance at 260 nm and 230 nm (A260 / 230) to provide purity assessment for isolated pDNA as measured with the NanodropTM8000.

[0031] FIG. 3 demonstrates reduction in endotoxin levels in presence and absence of polymyxin B sulfate.

[0032] FIG.4 provides data demonstrating that polymyxin B sulfate shows 98-99% reduction in endotoxin concentration compared to untreated control samples. DESCRIPTION OF VARIOUS EMBODIMENTS

[0033] It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not intended to limit the scope of the current teachings. In this application, the use of the singular includes the plural unless specifically stated otherwise. The use of "comprise", “contain”, and "include", or modifications of those root words, for example but not limited to, “comprises”, “contained”, and “including”, are not intended to be limiting. Use of “or” means “and / or” unless stated otherwise. The term “and / or” means that the terms before and after can be taken together or separately. For illustration purposes, but not as a limitation, “X and / or Y” can mean “X” or “Y” or “X and Y”.

[0034] Whenever a range of values is provided herein, the range is meant to include the starting value and the ending value and a value or value range there between unless otherwiseAttorney Docket No. TP387448WO1 specifically stated. For example, “from 0.2 to 0.5” means 0.2, 0.3, 0.4, 0.5; ranges there between such as 0.2-0.3, 0.3 – 0.4, 0.2 – 0.4; increments there between such as 0.25, 0.35, 0.225, 0.335, 0.49; increment ranges there between such as 0.26 – 0.39; and the like.

[0035] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described in any way. All literature and similar materials cited in this application including, but not limited to, patents, patent applications, articles, books, treatises, and internet web pages, regardless of the format of such literature and similar materials, are expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated literature and similar materials defines or uses a term in such a way that it contradicts that term’s definition in this application, this application controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

[0036] The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed items preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, AAABCCCC, CBBAAA, CABABB, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0037] Certain trademarked products are cited by teachings herein with reference to surfactants. Generic descriptions for such products are as follows: TRITON X-100™, octylphenol ethoxylate having an average of 9.5 ethoxylate groups (Dow Chemical Company Product Information, Form No. 119-01882, JMS1206); TRITON X-114™, octylphenol ethoxylate having an average of 7.5 ethoxylate groups (Dow Chemical Company Product Information, Form No. 119-01884, JMS1206); NONIDET P-40™, octylphenolpoly(ethyleneglycolether (Roche Diagnostics GmbH, Catalog No. 11 332 473Attorney Docket No. TP387448WO1 001, July 2005); and THESIT™, dodecyl alcohol polyoxyethylene ether (IUPAC Name 2- dodecoxyethanol; CAS Number 9002-92-0; Chemical Formula C14H30O2). Methods of Purifying Nucleic Acids

[0038] Nucleic Acids: Provided herein are compositions and methods for preparing nucleic acids from samples. Nucleic acids that can be prepared according to the disclosure provided herein include, for example, are single-stranded or double-stranded or double-stranded RNA or DNA, RNA / DNA hybrids, DNA fragments, oligonucleotides, genomic DNA, cDNA, PCR DNA, linear DNA, RNA, ribozymes, aptamers, and chemically synthesized nucleic acids, amplified DNA or RNA, BACs, or, in particular, plasmid DNA (“pDNA”). The size of the nucleic acids can be between 6 bp to about 15,000 bp. In preferred embodiments, the nucleic acids are pDNA, e.g. with a size of about 0.2 kb to about 2 Mb.

[0039] Samples: The term “sample,” as used herein, refers to an in vitro cell, cell culture or the like that contains nucleic acids. Samples useful in the embodiments provided herein can include cell cultures, e.g., of microorganisms or fungi that comprise nucleic acids (e.g., DNA, RNA, or the like, including pDNA), or environmental samples, or the like. For example, the samples can include cell cultures of Gram-negative microorganisms such as Escherichia coli that harbor pDNA.

[0040] The compositions and methods provided herein are useful for preparation of nucleic acids, e.g., pDNA from samples containing, e.g., from one cell up to about 5 x 1012cells per sample or any range therebetween. For example, the compositions and methods provided herein can be used to isolated pDNA from cultures of E. coli grown in nutrient broth to optical densities between about 0.01 to about 6.0. When the sample is a cell culture, e.g., of microorganisms, the culture medium preferably contains less than about 5 g / salt / L. The skilled artisan will appreciate that many types of cell cultures can be used as samples in the methods and compositions provided herein, including, for example, cultures of E. coli grown in LB broth, Terrific Broth, and the like.Attorney Docket No. TP387448WO1

[0041] The amount of sample processed can vary from about 50 mL to, e.g., less than 5 μL. In some embodiments, the samples include approximately 1.0 x 104- 9.0 x 1010cells / mL, such as E. coli cells.

[0042] Some of the methods provided herein include a first step of isolating microorganisms from a sample for further processing. For example, a liquid culture or an environmental sample containing a microorganism harboring nucleic acids can be treated with a solid support that binds to the microorganisms. As such, the solid support can be used to concentrate cells (e.g., microorganism(s)) in the sample, while at the same time purifying the cells (e.g., microorganism(s)) from the sample components (e.g., culture media). Solid supports useful for separating and concentrating cells (e.g., microorganism(s)) are described elsewhere herein.

[0043] `The samples can be provided in a minimal volume, e.g., less than 25 μL, preferably less than 10 μL (e.g., 5 μL or less). Preferably, the volume of lysis buffer used to contact the sample is more than 5-fold, e.g., 10-fold the volume of the sample.

[0044] The method provided for extracting a nucleic acid from a sample containing a nucleic acid wherein the method comprises: contacting the sample with said solid phase at a first pH at which the solid phase has a positive charge and will bind negatively charged nucleic acid; and then releasing the nucleic acid at a higher pH at which the solid phase possesses a neutral, negative or less positive charge than at the first pH. Furthermore, the solid phase will possess an overall positive charge, that is the sum of all positive and negative charges on the solid phase as a whole is positive.

[0045] The cell-bound (e.g., microorganism-bound) solid phase can be concentrated, e.g., by allowing the solid phase to settle, centrifugation, magnetic separation, or the like. Preferably, the cell-bound (e.g., microorganism-bound) solid phase is magnetic (e.g., paramagnetic or superparamagnetic), and can be separated from the rest of the sample by allowing the solid phase to bind to a magnet. Preferably, the solid phase is paramagnetic or superparamagnetic, and the beads can be easily released transferred to a different vessel (e.g., well or the like), containing a lysis / resuspension buffer as described elsewhere herein. The cells can be incubated with the lysis / resuspension buffer for a period of time, which can range from 1 minute to several hours, depending upon the incubation temperature. For example, the cellsAttorney Docket No. TP387448WO1 can be incubated for a period of time between 1 minute and 2 hours, at about 16 °C to 28 °C. For example, lysis mixtures can be incubated at 16 °C to 28 °C for 2 minutes to about 60 minutes, about 2 minutes to about 20 minutes, about 3 minutes to about 15 minutes, about 4 minutes to about 10 minutes or about 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes 34 minutes 35 minutes 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes 42 minutes 43 minutes, 44 minutes 45 minutes, 46 minutes 47 minutes, 48 minutes 49 minutes, 50 minutes, 51 minutes, 52 minutes 53 minutes 54 minutes 55 minutes 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, or longer, or any time in between. Alternatively, lysis mixtures can be held on ice, or incubated at 4 °C for 15 minutes to 12 hours or longer, e.g., 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer, or any time in between.

[0046] Lysis buffer: Lysis buffer composition provided herein comprises a denaturing agent. The lysis buffer provided herein can include a denaturing agent such as urea, thiourea, sodium deoxycholate, (cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), N-lauryl sarcosine (NLS), sodium hydroxide, potassium hydroxide, lithium hydroxide, barium hydroxide cesium hydroxide strontium hydroxide, rubidium hydroxide, or any combination thereof, at pH range from about 11.5 to about 14.0 for a range of temperatures such as 5 °C to 40 °C.

[0047] The lysis buffer used in the method of preparing endotoxin-free plasmid DNA (pDNA) can be provided as a stock solution at a concentration of about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold up to 100-fold or any concentrations in between. In some embodiments, the method of preparing endotoxin-free plasmid DNA (pDNA) comprises a step of lysing the microorganism wherein the lysis buffer comprises a denaturing agent such as sodium deoxycholate, (cetyltrimethylammonium bromide (CTAB), sodium dodecyl sulfate (SDS), lithium dodecyl sulfate (LDS), or N-lauryl sarcosine (NLS) in a concentration range from about 3 % w / v to about 8 % w / v relative to the total buffer mixtureAttorney Docket No. TP387448WO1 used in the method, e.g., 3 % w / v, 3.3 % w / v, 3.6 % w / v, 3.9 % w / v, 4.2 % w / v, 4.5 % w / v, 5 % w / v, 5.5% w / v, 6.0% w / v, 7.0 % w / v, 8 % w / v, or any value in between. In some embodiments, the method of preparing endotoxin-free plasmid DNA (pDNA) comprises a step of lysing the microorganism wherein the lysis buffer comprises a denaturing agent such as sodium hydroxide, potassium hydroxide, lithium hydroxide, barium hydroxide cesium hydroxide strontium hydroxide, or rubidium hydroxide in a concentration range from about 0.25 N to about 0.75 N relative to the total buffer mixture used in the method, e.g., 0.25 N, 0.3 N, 0.4 N, 0.5 N, 0.6 N, 0.7 N, 0.75 N, or any value in between.

[0048] In some embodiments, the pH of the lysis buffer composition is, or is about, or is no more than, or is no more than about, or is no less than, or is no less than about 11.5, 11.6, 11.7, 11.8, 11.9, 12.0, 12.1, 12.2, 12.3, 12.4, 12.5, 12.6, 12.7, 12.8, 12.9, 13.0, 13.1, 13.2, 13.3, 13.4, 13.5, 13.7, 13.8, 13.9, 14.0, or a range between any of these values. In some embodiments, the lysis buffer composition is at a pH from about 11.8 to about 14.0. In some embodiments, the lysis buffer composition is at a pH from about 12.0 to about 14.0. In some embodiments, the lysis buffer composition is at a pH from about 11.5 to about 12.5. In some embodiments, the lysis buffer composition is at a pH from about 11.8 to about 12.8. In some embodiments, the lysis buffer composition is at a pH of about 12.0. In some embodiments, the lysis buffer composition is at a pH of about 12.5.

[0049] Resuspension buffer: Resuspension buffer composition provided herein includes EDTA, HCl, tris-HCl, tris, arginine, glycine, ammonium formate, sodium citrate, or any combination thereof, at pH range from about 8.0 to about 10.0 for a range of temperatures such as 5 °C to 40 °C. In some embodiments, the pH of the resuspension buffer composition is, or is about, or is no more than, or is no more than about, or is no less than, or is no less than about 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, 9.6, 9.7, 9.8, 9.9, 10.0, or a range between any of these values. In some embodiments, the resuspension buffer composition is at a pH from about 8.0 to about 9.0. In some embodiments, the resuspension buffer composition is at a pH from about 8.2 to about 8.8. In some embodiments, the resuspension buffer composition is at a pH from about 8.4 to about 8.6. In some embodiments, the resuspension buffer composition is at a pH of about 8.4. In some embodiments, the resuspension buffer composition is at a pH of about 8.5.Attorney Docket No. TP387448WO1

[0050] The resuspension buffer used in the method of preparing endotoxin-free plasmid DNA (pDNA) can be provided as a stock solution at a concentration of about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold up to 100-fold or any concentrations in between. In some embodiments, the method of preparing endotoxin-free plasmid DNA (pDNA) comprises a step of incubating the microorganism-bound first solid phase with a resuspension buffer comprising a buffering agent in a concentration range from about 0.15 M to about 0.35 M relative to the total buffer mixture used in the method, e.g., 0.15 M, 0.17 M, 0.19 M, 0.21 M, 0.23 M, 0.25 M, 0.27 M, 0.29 M, 0.31 M, 0.33 M, 0.35 M, or any value in between. In some embodiments, the method of preparing endotoxin-free plasmid DNA (pDNA) comprises a step of incubating the microorganism-bound first solid phase with a resuspension buffer wherein the resuspension buffer further comprises another buffer agent in a concentration range from about 0.25 M to about 0.75 M relative to the total buffer mixture used in the method, e.g., 0.25 M, 0.3 M, 0.4 M, 0.5 M, 0.6 M, 0.7 M, 0.75 M, or any value in between. Preferably, the cationic detergent is present in a concentration of about 10% in the resuspension buffer, and about 5% in the lysis / resuspension buffer.

[0051] Lysis / Resuspension Buffer: The lysis and resuspension buffers described herein above can be combined, i.e., they can be provided as a single lysis / resuspension buffer. Preferably, the resuspension buffer contains an RNA-degradation component. The skilled artisan will appreciate that various RNA-degradation components (e.g., enzymatic, small molecule, etc.) are known, and can be used in the embodiments provided herein. For example, in some embodiments, the RNA-degradation component can be enzymatic, e.g., an RNase. In some embodiments, the RNA-degradation component can be a small molecule, e.g.,

[0052] The lysate obtained after separating the first solid phase is incubated with a composition for endotoxin removal for a period of time, which can range from 1 minute to several hours, depending upon the incubation temperature. For example, the lysate can be incubated for a period of time between 1 minute and 2 hours, at about 16 °C to 28 °C. For example, lysis mixtures can be incubated at 16 °C to 28 °C for 2 minutes to about 60 minutes, about 2 minutes to about 20 minutes, about 3 minutes to about 15 minutes, about 4 minutes to about 10 minutes or about 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes 17 minutes, 18Attorney Docket No. TP387448WO1 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes 34 minutes 35 minutes 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes 42 minutes 43 minutes, 44 minutes 45 minutes, 46 minutes 47 minutes, 48 minutes 49 minutes, 50 minutes, 51 minutes, 52 minutes 53 minutes 54 minutes 55 minutes 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, or longer, or any time in between. Alternatively, lysis mixtures can be held on ice, or incubated at 4 °C for 15 minutes to 12 hours or longer, e.g., 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer, or any time in between.

[0053] The endotoxin free lysate is contacted with a precipitation buffer and a second solid phase to produce a binding mixture, wherein the binding mixture is incubated for a period of time, which can range from 1 minute to several hours, depending upon the incubation temperature. For example, the binding mixture can be incubated for a period of time between 1 minute and 2 hours, at about 16 °C to 28 °C. For example, lysis mixtures can be incubated at 16 °C to 28 °C for 2 minutes to about 60 minutes, about 2 minutes to about 20 minutes, about 3 minutes to about 15 minutes, about 4 minutes to about 10 minutes or about 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes 34 minutes 35 minutes 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes 42 minutes 43 minutes, 44 minutes 45 minutes, 46 minutes 47 minutes, 48 minutes 49 minutes, 50 minutes, 51 minutes, 52 minutes 53 minutes 54 minutes 55 minutes 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, or longer, or any time in between. Alternatively, lysis mixtures can be held on ice, or incubated at 4 °C for 15 minutes to 12 hours or longer, e.g., 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer, or any time in between.

[0054] Precipitation buffer: Precipitation buffer provided herein includes sodium citrate, potassium acetate (KOAc), potassium citrate, NaCl, lithium acetate (LiOAc), sodium acetateAttorney Docket No. TP387448WO1 (NaOAc), lactic acid, malic acid, succinic acid, KCl, LiCl, HCl, or any combination thereof, at pH range from about 3.5 to about 5.5 for a range of temperatures such as 5 °C to 40 °C. In some embodiments, the precipitation buffer provided herein includes short chain alcohols such as methanol, ethanol, isopropanol, 2-methyl-1,3-propanediol (MPD), or any combination thereof. In some embodiments, the pH of the precipitation buffer composition is, or is about, or is no more than, or is no more than about, or is no less than, or is no less than about 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, or a range between any of these values. In some embodiments, the precipitation buffer composition is at a pH from about 3.7 to about 4.5. In some embodiments, the precipitation buffer composition is at a pH from about 3.8 to about 4.3. In some embodiments, the precipitation buffer composition is at a pH from about 3.9 to about 4.1. In some embodiments, the precipitation buffer composition is at a pH of about 3.9. In some embodiments, the precipitation buffer composition is at a pH of about 4.0.

[0055] The precipitation buffer used in the method of preparing endotoxin-free plasmid DNA (pDNA) can be provided as a stock solution at a concentration of about 1-fold, 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10- fold up to 100-fold or any concentrations in between. In some embodiments, the method of preparing endotoxin-free plasmid DNA (pDNA) comprises a step of incubating the microorganism-bound first solid phase with a precipitation buffer comprising potassium acetate (KOAc), potassium citrate, lithium acetate (LiOAc), sodium acetate (NaOAc), lactic acid, or malic acid in a concentration range from about 0.1 M to about 0.4 M relative to the total buffer mixture used in the method, e.g., 0.1 M, 0.15 M, 0.17 M, 0.19 M, 0.21 M, 0.23 M, 0.25 M, 0.27 M, 0.29 M, 0.31 M, 0.33 M, 0.35 M, 0.37 M, 0.4 M, or any value in between.

[0056] In some embodiments, the method of preparing endotoxin-free plasmid DNA (pDNA) comprises a step of incubating the microorganism-bound first solid phase with a precipitation buffer comprising KCl, LiCl, or NaCl, in a concentration range from about 0.1 M to about 0.4 M relative to the total buffer mixture used in the method, e.g., 0.1 M, 0.15 M, 0.17 M, 0.19 M, 0.21 M, 0.23 M, 0.25 M, 0.27 M, 0.29 M, 0.31 M, 0.33 M, 0.35 M, 0.37 M, 0.4 M, or any value in between.Attorney Docket No. TP387448WO1

[0057] Wash buffers: Wash buffers provided herein includes ammonium sulfate, ammonium persulfate, water, nuclease free water, ultrapure water, or any combination thereof. In some embodiments, the wash buffer comprises nuclease free water. In some embodiments, the wash buffer comprises ultrapure water. In some embodiments, the wash buffer comprises ammonium sulfate in concentrations ranging from about 0.25 M to about 0.50 M. In some embodiments, the wash buffer comprises ammonium sulfate in concentrations ranging from about 0.28 M to about 0.48 M. In some embodiments, the wash buffer comprises ammonium sulfate in concentrations ranging from about 0.30 M to about 0.45 M. In some embodiments, the wash buffer comprises ammonium sulfate in concentrations ranging from about 0.32 M to about 0.42 M. In some embodiments, the wash buffer comprises ammonium sulfate in concentrations ranging from about 0.32 M to about 0.38 M. In some embodiments, the wash buffer comprises ammonium sulfate in concentrations ranging from about 0.34 M to about 0.36 M. In certain embodiments, the ammonium sulfate is present at about 0.25 M, 0.28 M, 0.30 M, 0.32 M, 0.34 M, 0.35 M, 0.36 M, 0.38 M, 0.40 M, 0.42 M, 0.44 M, 0.46 M, 0.48 M, 0.50 M, or any range of concentrations there between.

[0058] The second solid phase is contacted with an elution buffer at a temperature, for a period of time, which can range from 1 minute to several hours, depending upon the incubation temperature. For example, the binding mixture can be incubated for a period of time between 1 minute and 2 hours, at about 16 °C to 28 °C. For example, lysis mixtures can be incubated at 16 °C to 28 °C for 2 minutes to about 60 minutes, about 2 minutes to about 20 minutes, about 3 minutes to about 15 minutes, about 4 minutes to about 10 minutes or about 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes 17 minutes, 18 minutes, 19 minutes, 20 minutes, 21 minutes, 22 minutes, 23 minutes, 24 minutes, 25 minutes, 26 minutes, 27 minutes, 28 minutes, 29 minutes, 30 minutes, 31 minutes, 32 minutes, 33 minutes 34 minutes 35 minutes 36 minutes, 37 minutes, 38 minutes, 39 minutes, 40 minutes, 41 minutes 42 minutes 43 minutes, 44 minutes 45 minutes, 46 minutes 47 minutes, 48 minutes 49 minutes, 50 minutes, 51 minutes, 52 minutes 53 minutes 54 minutes 55 minutes 56 minutes, 57 minutes, 58 minutes, 59 minutes, 60 minutes, or longer, or any time in between. Alternatively, lysis mixtures can be held on ice, or incubated at 4 °C for 15 minutes to 12 hours or longer, e.g., 30 minutes, 45Attorney Docket No. TP387448WO1 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, or longer, or any time in between.

[0059] Elution buffer: Elution buffer provided herein includes tris-HCl, NaOH, tris, or any combination thereof, at pH range from about 8.0 to about 9.5 for a range of temperatures such as 5 °C to 40 °C. In some embodiments, the pH of the elution buffer composition is, or is about, or is no more than, or is no more than about, or is no less than, or is no less than about 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0, 9.1, 9.2, 9.3, 9.4, 9.5, or a range between any of these values. In some embodiments, the elution buffer composition is at a pH from about 8.0 to about 9.0. In some embodiments, the elution buffer composition is at a pH from about 8.2 to about 8.8. In some embodiments, the elution buffer composition is at a pH from about 8.4 to about 8.6. In some embodiments, the elution buffer composition is at a pH of about 8.4. In some embodiments, the elution buffer composition is at a pH of about 8.5.

[0060] Temperature: The methods provided herein include incubation at a temperature, which is preferably between about 15 °C to 40 °C, or about 16 °C to 28 °C or about 19 °C to 26 °C, or about 19 °C to 25 °C, or about 22 °C to 25 °C, or at ambient temperature, or about 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C, 34 °C, 35 °C, 36 °C, 37 °C, 38 °C, 39 °C, or 40 °C. Preferably, during all incubation steps the mixtures remains at substantially the same temperature during the incubation time. “Substantially the same temperature” generally refers to an isothermal process of holding the temperature relatively constant during the incubation time, for certain embodiments described herein, means ambient temperature which temperature may change during the day or from lab to lab. An isothermal process is particularly amenable for high throughput analyses.

[0061] Endotoxin removal Buffer (ERB): Provided herein are endotoxin removal buffers. include an alkali or alkaline earth metal salt, a non-ionic detergent, a polymyxin, and a buffering agent. The composition provided herein can include a buffering agent, such as N- 2-acetamido-2-aminoethanesulfonic acid (ACES), N-2-acetamido-2-iminodiacetic acid (ADA), 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid (AMPSO), N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES), 4-cyclohexylamino-1-butaneAttorney Docket No. TP387448WO1 sulfonic acid (CABS), 3-cyclohexylamino-1-propane sulfonic acid (CAPS), 3- cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO), 2-N- cyclohexylaminoethanesulfonic acid (CHES), 3-N,N-bis-2-hydroxyethylamino-2- hydroxypropanesulfonic acid (DIPSO), N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS or HEPPS), N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS), 4-N- morpholinobutanesulfonic acid (MOBS), 3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris), hydroxyethylpiperazine ethane sulfonic acid (HEPES), morpholinoethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), [tris(hydroxymethyl)methylamino] propanesulfonic acid (TAPS), N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS), N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS), 3-N- trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO), N- trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES), N-(2-acetamido)iminodiacetic acid (ADA), Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), N- trishydroxymethylmethylglycine (TRICINE), or any combination thereof, at pH 4.5 to 6.5 for a range of temperatures such as 5 °C to 40 °C. In some embodiments, the pH of the endotoxin removal buffer composition is, or is about, or is no more than, or is no more than about, or is no less than, or is no less than about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, or a range between any of these values. In some embodiments, the endotoxin removal buffer composition is at a pH from about 4.5 to about 6.0. In some embodiments, the endotoxin removal buffer composition is at a pH from about 4.5 to about 5.5. In some embodiments, the endotoxin removal buffer composition is at a pH from about 4.7 to about 5.8. In some embodiments, the endotoxin removal buffer composition is at a pH from about 4.9 to about 5.7. In some embodiments, the endotoxin removal buffer composition is at a pH from about 4.9 to about 5.5. In some embodiments, the endotoxin removal buffer composition is at a pH from about 4.9 to about 5.3. In some embodiments, the endotoxin removal buffer composition is at a pH from about 4.9 to about 5.1. In some embodiments, the endotoxin removal buffer composition is at a pH of about 4.9. In some embodiments, the endotoxin removal buffer composition is at a pH of about 5.0. In some embodiments, the endotoxin removal buffer composition is at a pH of about 5.1. In someAttorney Docket No. TP387448WO1 embodiments, the endotoxin removal buffer composition is at a pH of about 5.2. In some embodiments, the endotoxin removal buffer composition is at a pH of about 5.3.

[0062] Detergents: In embodiments provided herein, the compositions comprise a detergent. Preferably, the detergent is provided at a concentration that has low or no emission at the emission wavelengths of commonly used RNA-or DNA-detectable labels (e.g., between about 300 nm and 750 nm), and wherein the concentration is lysis-effective. Preferably, the detergent is REACH compliant.

[0063] Various non-ionic detergents or any combinations thereof are known in the art and can be used in the compositions provided herein. For example, the compositions can include a non-ionic detergent, such as for example, one or more of Tergitol 15-S-7, Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, Tergitol NP-50, Tergitol NP-30, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol 26- 7, Tergitol 15-S-20, Tergitol NP-70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S-5, Pluronic F-127, Synperonic®F 108, Synperonic®PE P105, Ecosurf™ EH-9, Ecosurf™SA-4, Ecosurf™SA-9, Ecosurf™EH-6, Ecosurf™EH-3, Saponin, Ecosurf™SA-7, Poloxamer 188, Tween®80, Tween®85, Tween®40, Tween®20, Tween®60, Tween®65, Triton X-45, Triton X-100, Triton X-114, Triton X-102, Triton X-165, Triton X-305, Triton X-705, Triton™X-405, Triton™X-405, reduced, Triton™X-100 reduced, Triton™N-101, reduced, Triton™CG-110, Brij®35, Brij®58, Brij®L23, Brij®S10, BRIJ®O20, Brij®S 100, Brij®O10, Brij®S20, Brij®C10, Brij®L4, Brij®93, SP Brij®S2 MBAL, Digitonin, MERPOL®A, MERPOL®HCS, MERPOL®SH, MERPOL®SE, Elugent, Octyl β-D-glucopyranoside, n-Dodecyl β-D-maltoside, Decyl β-D-maltopyranoside, n-Octyl β-D- maltoside, Decyl β-D-glucopyranoside, Octyl α-D-glucopyranoside, Hexyl β-D- glucopyranoside, Nonyl β-D-maltoside, IGEPAL®CA-630, IGEPAL®CO-520, IGEPAL®CO-630, IGEPAL®CA-720, IGEPAL®CO-890, Octyl-beta-Glucoside, Octylthio Glucoside, cocoamide monoethanolamine (Cocamide MEA), cocamide diethanolamine (Cocamide DEA), or any combination thereof.

[0064] By way of example, an endotoxin removal composition provided herein includes a non-ionic surfactant is selected from the group consisting of Tergitol 15-S-7, Tergitol 15-S-9,Attorney Docket No. TP387448WO1 Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, Tergitol NP-50, Ecosurf™ EH-9, Ecosurf™SA-4, Ecosurf™SA-9, Ecosurf™EH-6, Ecosurf™EH-3, Saponin, Ecosurf™SA-7, Poloxamer 188, Tween®80, Tween®85, Tween®40, Tween®20, Tween®60, Tween®65, Triton X-45, Triton X-100, Triton X-114, Triton X-102, Triton X-165, Triton X-305, Triton X-705, Triton™X-405, Tergitol NP-30, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S-20, Tergitol NP-70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S-5, or any combination thereof.

[0065] Concentrations of non-ionic detergents range from 0.001% to 25% (v / v) or more. Accordingly, an effective concentration of non-ionic detergent can be from 0.05% to 25%, e.g., range from 0.05% to 22.5%, from 0.05% to 20%, from 0.05% to 17.5%, from 0.05% to 15%, from 0.05% to 12.5%, from 0.05% to 10.5%, from 0.1% to 10.5%, from 0.5% to 10.5%, from 0.5% to 7.5%, from 0.5% to 5.5%, from 1% to 10%, from 3% to 10%, from 5% to 10%, or more.

[0066] In some embodiments, the method of preparing endotoxin-free plasmid DNA (pDNA) comprises a step of adding a second solid phase and an endotoxin removal buffer to the pDNA-enriched solution comprising a non-ionic detergent in a concentration range from about 0.5 % w / v to 1.3 % w / v relative to the total buffer mixture used in the method, e.g.0.5 % w / v, 0.6 % w / v, 0.7 % w / v, 0.8 % w / v, 0.9 % w / v, 1.0 % w / v, 1.1 % w / v, 1.2 % w / v, 1.3 % w / v, or any value in between.

[0067] Salts: Various alkali or alkaline earth metal salts or any combinations thereof are known in the art and can be used in the compositions provided herein. For example, the compositions can include an alkali or alkaline earth metal salt, such as for example, one or more of LiCl, NaCl, KCl, MgCl2, BaCl2, CaCl2, or any combination thereof.

[0068] Salts: In some embodiments, the endotoxin removal composition includes one or more salts, such as alkali metal salts (e.g. sodium, potassium, and / or lithium salts) or alkaline earth metal salts (e.g, calcium barium, and / or magnesium salts). For example, compositions provided herein can include salt in concentrations ranging from about 0.05 M to about 2.0 M. In some embodiments, the salt is present in the compositions in concentrations ranging fromAttorney Docket No. TP387448WO1 about 0.05 M to about 1.5 M. In some embodiments, the salt is present in the compositions in concentrations ranging from about 0.1 M to about 1.5 M. In some embodiments, the salt is present in the compositions in concentrations ranging from about 0.2 M to about 1.5 M. In some embodiments, the salt is present in the compositions in concentrations ranging from about 0.3 M to about 1.25 M. In some embodiments, the salt is present in the compositions in concentrations ranging from about 0.4 M to about 1.35 M. In some embodiments, the salt is present in the compositions in concentrations ranging from about 0.5 M to about 1.4 M. In some embodiments, the salt is present in the compositions in concentrations ranging from about 0.5 M to about 1.2 M. In some embodiments, the salt is present in the compositions in concentrations ranging from about 0.5 M to about 1.0 M. In some embodiments, the salt is present in the compositions in concentrations ranging from about 0.6 M to about 1.0 M. In certain embodiments, the alkali salt or alkaline earth metal salt is present at about 0.05 M, 0.1 M, 0.15 M, 0.2 M, 0.25 M, 0.3 M, 0.35 M, 0.4 M, 0.45 M, 0.5 M, 0.55 M, 0.6 M, 0.65 M, 0.7 M, 0.75 M, 0.8 M, 0.85 M, 0.9 M, 0.95 M, 1.0 M, 1.15 M, 1.2 M, 1.25 M, 1.3 M, 1.35 M, 1.4 M, 1.45 M, 1.5 M, 1.55 M, 1.6 M, 1.65 M, 1.7 M, 1.75 M, 1.8 M, 1.85 M, 1.9 M, 1.95 M, 2.0 M, or any range of concentrations there between.

[0069] In some embodiments, the method of preparing endotoxin-free plasmid DNA (pDNA) comprises a step of adding a second solid phase and an endotoxin removal buffer to the pDNA-enriched solution comprising a salt such as one or more of LiCl, NaCl, KCl, MgCl2, BaCl2, CaCl2, or any combination thereof, in a concentration range from about 0.05 M to about 0.15 M relative to the total buffer mixture used in the method, e.g., 0.05 M, 0.055 M, 0.06 M, 0.065 M, 0.07 M, 0.075 M, 0.08 M, 0.085 M, 0.09 M, 0.1 M, 0.125 M, 0.15 M, or any value in between.

[0070] Buffering agents: Various buffering agents or any combinations thereof are known in the art and can be used in the compositions provided herein. For example, the compositions can include a buffering agent, wherein the wherein the buffering agent is selected from N-2- acetamido-2-aminoethanesulfonic acid (ACES), N-2-acetamido-2-iminodiacetic acid (ADA), 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid (AMPSO), N,N-bis2- hydroxyethyl-2-aminoethanesulfonic acid (BES), 4-cyclohexylamino-1-butane sulfonic acid (CABS), 3-cyclohexylamino-1-propane sulfonic acid (CAPS), 3-cyclohexylamino-2-Attorney Docket No. TP387448WO1 hydroxy-1-propane sulfonic acid (CAPSO), 2-N-cyclohexylaminoethanesulfonic acid (CHES), 3-N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO), N-2- hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS or HEPPS), N-2- hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS), 4-N-morpholinobutanesulfonic acid (MOBS), 3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO), Bis-Tris, hydroxyethylpiperazine ethane sulfonic acid (HEPES), morpholinoethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), [tris(hydroxymethyl)methylamino] propanesulfonic acid (TAPS), N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS), N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS), 3-N- trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO), N- trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES), N-(2-acetamido)iminodiacetic acid (ADA), Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), Tris-Hydrochloride (Tris- HCl), N-trishydroxymethylmethylglycine (TRICINE), or any combination thereof.

[0071] Buffering agents: In some embodiments, the endotoxin removal composition includes one or more buffering agents. For example, compositions provided herein can include buffering agents in concentrations ranging from 7.5 mM to 120 mM. In some embodiments, the buffering agent is present in the compositions in concentrations ranging from about 7.5 mM to about 110 mM. In some embodiments, the buffering agent is present in the compositions in concentrations ranging from about 7.5 mM to about 105 mM. In some embodiments, the buffering agent is present in the compositions in concentrations ranging from about 8.5 mM to about 105 mM. In some embodiments, the buffering agent is present in the compositions in concentrations ranging from about 9.0 mM to about 105 mM. In some embodiments, the buffering agent is present in the compositions in concentrations ranging from about 10 mM to about 105 mM. In some embodiments, the buffering agent is present in the compositions in concentrations ranging from about 10 mM to about 100 mM. In some embodiments, the buffering agent is present in the compositions in concentrations ranging from about 10 mM to about 90 mM. In some embodiments, the buffering agent is present in the compositions in concentrations ranging from about 25 mM to about 80 mM. In some embodiments, the buffering agent is present in the compositions in concentrations ranging from about 35 mM to about 70 mM. In some embodiments, the buffering agent is present in the compositions in concentrations ranging from about 40 mM to about 60 mM. In someAttorney Docket No. TP387448WO1 embodiments, the buffering agent is present in the compositions in concentrations ranging from about 45 mM to about 55 mM. In certain embodiments, the buffering agent is present at about 7.5 mM, 10.0 mM, 15.0 mM, 20.0 mM, 25.0 mM, 30.0 mM, 35.0 mM, 40.0 mM, 45.0 mM, 50.0 mM, 55.0 mM, 60.0 mM, 65.0 mM, 70.0 mM, 75.0 mM, 80.0 mM, 85.0 mM, 90.0 mM, 95.0 mM, 100.0 mM, 105.0 mM, 110.0 mM, 115.0 mM, 120.0 mM, or any range of concentrations there between.

[0072] In some embodiments, the method of preparing endotoxin-free plasmid DNA (pDNA) comprises a step of adding a second solid phase and an endotoxin removal buffer to the pDNA-enriched solution comprising a buffering agent in a concentration range from about 0.001 M to about 0.01 M relative to the total buffer mixture used in the method, e.g., 0.001 M, 0.002 M, 0.003 M, 0.004 M, 0.005 M, 0.006 M, 0.007 M, 0.008 M, 0.009 M, 0.01 M, or any value in between.

[0073] Polymyxins: Polymyxins are antibiotics. Polymyxins B and E (also known as colistin) are used in the treatment of Gram-negative bacterial infections. It is well demonstrated that the bacterial cytoplasmic membrane is partially damaged upon polymyxin exposure and that components of the cytoplasmic membrane are released in fibrous forms through crevices. By binding to the Lipid A component of bacterial cell wall lipopolysaccharide (LPS, or endotoxin), polymyxins also may interfere with the biologic function of LPS. In some embodiments, the endotoxin removal composition includes a polymyxin, wherein the polymyxin is selected from the group consisting of polymyxin-B and polymyxin-E. For example, compositions provided herein can include a polymyxin in concentrations ranging from about 0.2 mg / mL to about 20.0 mg / mL. In some embodiments, the polymyxin is present in the compositions in concentrations ranging from about 0.2 mg / mL to about 17.5 mg / mL. In some embodiments, the polymyxin is present in the compositions in concentrations ranging from about 0.2 mg / mL to about 15.0 mg / mL. In some embodiments, the polymyxin is present in the compositions in concentrations ranging from about 0.5 mg / mL to about 15.0 mg / mL. In some embodiments, the polymyxin is present in the compositions in concentrations ranging from about 0.75 mg / mL to about 12.5 mg / mL. In some embodiments, the polymyxin is present in the compositions in concentrations ranging from about 0.9 mg / mL to about 10.5 mg / mL. In some embodiments, the polymyxin is present in the compositions inAttorney Docket No. TP387448WO1 concentrations ranging from about 2.0 mg / mL to about 10.0 mg / mL. In some embodiments, the polymyxin is present in the compositions in concentrations ranging from about 1.0 mg / mL to about 8.5 mg / mL. In some embodiments, the polymyxin is present in the compositions in concentrations ranging from about 2.0 mg / mL to about 8.5 mg / mL. In some embodiments, the polymyxin is present in the compositions in concentrations ranging from about 3.0 mg / mL to about 7.0 mg / mL. In some embodiments, the polymyxin is present in the compositions in concentrations ranging from about 3.5 mg / mL to about 7.0 mg / mL. In some embodiments, the polymyxin is present in the compositions in concentrations ranging from about 4.5 mg / mL to about 6.5 mg / mL. In certain embodiments, the polymyxin is present at about 0.2 mg / mL, 0.5 mg / mL, 0.75 mg / mL, 0.8 mg / mL, 0.85 mg / mL, 0.9 mg / mL, 1.0 mg / mL, 1.5 mg / mL, 2.0 mg / mL, 2.25 mg / mL, 2.5 mg / mL, 2.75 mg / mL, 3.0 mg / mL, 3.5 mg / mL, 4.0 mg / mL, 5.0 mg / mL, 5.5 mg / mL, 6.0 mg / mL, 6.5 mg / mL, 7.0 mg / mL, 7.5 mg / mL, 8.0 mg / mL, 8.5 mg / mL, 9.0 mg / mL, 9.5 mg / mL, 10.0 mg / mL, 10.5 mg / mL, 11.0 mg / mL, 11.5 mg / mL, 12.0 mg / mL, 12.5 mg / mL, 13.0 mg / mL, 13.5 mg / mL, 14.0 mg / mL, 14.5 mg / mL, 15.0 mg / mL, 15.5 mg / mL, 16.0 mg / mL, 17.0 mg / mL, 17.5 mg / mL, 18.0 mg / mL, 18.5 mg / mL, 19.0 mg / mL, 20.0 mg / mL, or any range of concentrations there between.

[0074] In some embodiments, the method of preparing endotoxin-free plasmid DNA (pDNA) comprises a step of adding a second solid phase and an endotoxin removal buffer to the pDNA-enriched solution comprising a polymyxin in a concentration range from about 0.02 % w / v to 0.08 % w / v relative to the total buffer mixture used in the method, e.g. 0.02 % w / v, 0.03 % w / v, 0.04 % w / v, 0.05 % w / v, 0.06 % w / v, 0.07 % w / v, 0.08 % w / v, or any value in between.

[0075] Solid Phase: Generally, the solid phase will possess an overall positive charge, that is the sum of all positive and negative charges on the solid phase as a whole is positive. It is possible (though not preferred), however, that the solid phase as a whole could be negatively charged but have areas of predominantly positive charge to which the nucleic acid can bind. Such solid phases are within the scope of the present disclosure.

[0076] In some embodiments, chemical species comprising ionizable groups can be immobilized onto the solid phases (for example, beads, particles, tubes, wells, probes,Attorney Docket No. TP387448WO1 dipsticks, pipette tips, slides, fibers, membranes, papers, celluloses, agaroses, glass or plastics) in a monomeric or polymeric form via adsorption, ionic, or covalent interactions, or by covalent attachment to a polymer backbone which is in turn immobilized onto the solid phase.

[0077] In some embodiments, the solid phase materials, especially beads and particles, may be magnetizable, magnetic, or paramagnetic. In some embodiments, the magnetic or paramagnetic beads can aid removal of the solid phase from a solution containing the released nucleic acid, prior to further processing or storage of the nucleic acids, RNA or DNA.

[0078] In some embodiments, the chemical species comprise a positively ionizable group (for example, a nitrogen atom), and at least one, but preferably more than one, electronegative group, for example, hydroxy, carboxy, carbonyl, phosphate or sulphonic acid group or C=C double bond, which is sufficiently close to the ionizable group, i.e., a nitrogen atom, to lower its pKa. It has been found that such molecules tend to have suitable pKa values for the extraction of nucleic acids (e.g. pDNA) under mild conditions according to the present disclosure. Nucleic acid will be bound to the solid phase at a pH below or roughly equal to the pKa, when the solid phase is positively charged, and will be released at a higher pH (usually above the pKa), when the solid phase is less positively charged, neutral, or negatively charged. The solid phase will change charge because of a change in charge on a positively ionizable group from positive to less positive or neutral, as the pH is increased in a range spanning or close to the pKa of the positively ionizable group. In some embodiments, the solid phase material may comprise an ionizable group having a pKa between about 3 and 9. In some embodiments, for positively ionizable groups, the pKa is more preferably at least about 4.5, 5.0, 5.5, 6.0, or 6.5 and / or at most about 7.0, 7.5, 8.0, or 8.5. In some embodiments, a particularly preferred pKa for a positively ionizable group is between about 5 and 8; even more preferred is a pKa between about 6.0 and 7.0, more preferably between about 6.5 and 7.0. In some embodiments, the pKa for negatively ionizable groups is preferably between about 3 and 7, more preferably between about 4 and 6, further preferably approximately at the pH at which it is desired to bind nucleic acid. In some embodiments, solid phase materials having more than one pKa value (e.g. having different ionisable groups), or combinations of materials having different pKa values, may also be suitable for use as solid phase inAttorney Docket No. TP387448WO1 accordance with the invention, provided that at a first (lower) pH the material(s) possess(es) a positive charge and that at a higher pH the charge is less positive, neutral or negative.

[0079] In particular, the nucleic acid binding step on the solid phase is carried out at a pH of below the pKa of the ionizable group, or (though this is not preferred) within about 1 pH unit above the pKa. In particular, the nucleic acid releasing step on the solid phase is carried out at a pH above the pKa of the ionizable group, preferably at a pH between 1 and 3 pH units above the pKa.

[0080] In some embodiments, the step of binding nucleic acid occurs under mild conditions, suitably at a pH of no less than 3.0, preferably no less than 3.5, 4.0, 4.5 or 5.0. In some embodiments, the nucleic acid releasing step is performed at a pH of no greater than about pH 10.5, more preferably no greater than about pH 10.0, 9.8, 9.6, 9.4, 9.2, 9.0, 8.9, 8.8, 8.7, 8.6 or 8.5. Also, depending on the pKa(s) of the solid phase, the releasing step may even be performed at lower pH values, such as 8.0, 7.5 or 7.0.

[0081] In some embodiments, the chemical species immobilized onto the solid phases, comprise a positively ionizable nitrogen atom, and at least one electronegative group (but more preferably more than one) wherein the electronegative group is separated from the ionizable nitrogen by no more than two atoms (usually carbon atoms). In some embodiments, hydroxyl groups are particularly preferred electronegative groups particularly when several hydroxyl groups are present, for example in polyhydroxylated amines, such as tris(hydroxymethyl)aminomethane (Tris; C(CH2OH)3—NH2) or bis-2- hydroxyethyliminotrishydroxymethylmethane (Bis-Tris; C(CH2OH)3—N(CH2CH2OH)2).

[0082] In some embodiments, polyhydroxylated amines, such as tris(hydroxymethyl)aminomethane (Tris; C(CH2OH)3—NH2) or bis-2- hydroxyethyliminotrishydroxymethylmethane (Bis-Tris; C(CH2OH)3—N(CH2CH2OH)2), lower the pKa of the nitrogen atom (for example, amine group from about 10 or 11 to a suitable value around neutral, i.e., pKa of about 7) and allow the species to remain soluble / hydrophilic above the pKa, when the nitrogen atom of the amine group loses its positive charge and provide a site for covalent linkage to a solid phase substrate, for example, a polycarboxylated polymer (such as polyacrylic acid or poly-methacrylic acid). In some embodiments,Attorney Docket No. TP387448WO1 polyhydroxylated amines, such as tris(hydroxymethyl)aminomethane (Tris; C(CH2OH)3— NH2) or bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris; C(CH2OH)3— N(CH2CH2OH)2) are uncharged at pH values suitable for the step of releasing the nucleic acids and at which procedures such as PCR are performed (typically pH ~ 8.5). In some embodiments, the preferred chemical species are having an ionizable nitrogen atom and at least 2, 3, 4, 5, or 6 hydroxyl groups.

[0083] In some embodiments, chemical species comprising ionizable groups can be immobilized onto the first and second solid phases, wherein the chemical species comprise a plurality of ionizable groups selected from the group consisting of N- trishydroxymethylmethylglycine (TRICINE), trishydroxymethylaminomethane (Tris), polyhydroxylated imidazoles, bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris), 1,3-bistrishydroxymethylmethylaminopropane (Bis-TrisPropane), N- trishydroxymethylmethylglycine (TRICINE), trishydroxymethylaminomethane (Tris), polyhydroxylated imidazoles, triethanolamine dimers and polymers, heterocyclic nitrogen- containing aromatic or aliphatic compounds and may be monomers, oligomers or polymers, such as morpholine-, pyrrole-, pyrrolidine-, pyridine-, pyridinol-, pyridone-, pyrroline-, pyrazole-, pyridazine-, pyrazine-, piperidone-, piperidine-, or piperazine-containing compounds.

[0084] Kits: A "kit," as used herein, refers to a combination of items for performing a sample preparation method as set forth herein. Kits provided herein can include a composition for endotoxin removal, as described herein above. By way of example, kits provided herein can include a buffer that includes an alkali or alkaline earth metal salt (e.g., LiCl, NaCl, KCl, MgCl2, BaCl2, and CaCl2); a non-ionic detergent (e.g., Tergitol 15-S-7, Tergitol 15-S-9, Tergitol 15-S-12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, Tergitol NP-50, Ecosurf™ EH-9, Ecosurf™SA-4, Ecosurf™SA-9, Ecosurf™EH-6, Ecosurf™EH-3, Saponin, Ecosurf™SA-7, Poloxamer 188, Tween®80, Tween®85, Tween®40, Tween®20, Tween®60, Tween®65, Triton X-45, Triton X-100, Triton X-114, Triton X-102, Triton X-165, Triton X-305, Triton X-705, Triton™X-405, Tergitol NP-30, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S-20, Tergitol NP-70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S-5, or any combinationAttorney Docket No. TP387448WO1 thereof); a polymyxin; and a buffering agent (e.g., N-2-hydroxyethylpiperazine-N-4- butanesulfonic acid (HEPBS), 4-N-morpholinobutanesulfonic acid (MOBS), 3-N- morpholino-2-hydroxypropanesulfonic acid (MOPSO), bis(2-hydroxyethyl)amino- tris(hydroxymethyl)methane (Bis-Tris), hydroxyethylpiperazine ethane sulfonic acid (HEPES), morpholinoethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), [tris(hydroxymethyl)methylamino] propanesulfonic acid (TAPS), N- trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS), N-trishydroxymethyl- methyl-3-aminopropanesulfonic acid (TAPS), 3-N-trishydroxymethyl-methylamino-2- hydroxypropanesulfonic acid (TAPSO), N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES), N-(2-acetamido)iminodiacetic acid (ADA), or any combination thereof). Components of kits may be packaged together or separately as desired for the processes described herein.

[0085] Kits can further include reagents for isolation of pDNA from a biological sample, such a lysis buffer, comprising a denaturing agent; a re-suspension buffer comprising a RNase; a precipitation buffer comprising an alkali metal salt; and an elution buffer comprising a buffering agent and an alkali metal hydroxide.

[0086] When components of a kit are provided in one and / or more liquid solutions, the liquid solution comprises an aqueous solution that can be a sterile aqueous solution. In some embodiments, the components of the kit can be provided as dried powder(s). When reagents and / or components are provided as a dry powder, the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent can also be provided in another container means. The container means will generally include at least one vial, test tube, flask, bottle, syringe and / or other container means, into which the solutions are placed, and in some embodiments, suitably aliquoted. The kits can also comprise a further container means for containing a sterile, pharmaceutically acceptable buffer and / or another diluent.

[0087] A kit can also include instructions for employing the kit components as well the use of any other reagent not included in the kit. Instructions can include variations that can be implemented.Attorney Docket No. TP387448WO1

[0088] Aspects of the present teachings can be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way. Example 1 Amine loaded on Magnetic Beads

[0089] 200 mg of carboxylated magnetic particles were treated with 160 mg of amino alcohol and 50 mg EDC in 0.1 M imidazole buffer, pH ~ 6. The resulting suspension was mixed at ambient conditions overnight. Following an overnight incubation, the magnetic particles were washed and used to isolate pDNA. Example 2 BisTris loaded on Magnetic Beads

[0075] 200 mg of polyacrylic acid coated magnetic particles were treated with 1340 mg of BisTris and 33 mg EDC in 0.1 M imidazole buffer pH ~ 6. The resulting suspension was mixed at ambient conditions overnight. Following an overnight incubation, the magnetic particles Example 3 Effect of Surfactant on Preparation of Samples for pDNA Analysis

[0076] Studies were conducted to assess the effectiveness of different non-ionic surfactants for the preparation of lysates for gene expression analysis by RT-qPCR. A buffer composition was prepared that consisted of 0.75 M NaCl, 0.05 M MOPS, and 10% of non-ionic surfactant. The non-ionic surfactants tested included: Tergitol 15-S-7, Tergitol 15-S-9, and Ecosurf™EH- 9. Studies were conducted to assess the effectiveness of different non-ionic surfactants for the preparation of lysates for plasmid DNA concentration analysis by Nanodrop 8000. A buffer composition was prepared that consisted of 0.75 M NaCl, 0.05 M MOPS, and 10% of non-ionic surfactant. The non-ionic surfactants tested included: Tergitol 15-S-7, Tergitol 15- S-9, and Ecosurf™ EH-9. E.coli strain TOP10 transformed with plasmid pSEAP2 was grown overnight for approximately 14-16 hours in LB broth shaking overnight at 225rpm in at 37◦C incubator. 1 mL of the E. coli culture was added to 24 individual wells of a 96-wellAttorney Docket No. TP387448WO1 KingFisher™ Deep well plate. The plate was processed on the KingFisher™ Apex system. 45 uL of buffers 1, 2, and 3 were added in addition to the previous buffer iteration made of 10% Triton X-100. As a control, no endotoxin removal buffer was added to four of the samples. Samples were quantified using the Nanodrop 8000.

[0077] Comparison of the yields in micrograms (ug) obtained from quantification revealed an improvement in yields for samples treated with endotoxin removal buffers, indicating all non- ionic buffers tested do not negatively impact yields in a mini-prep workflow. These samples also had purity ratios of wavelengths (nM) 260 / 230 and 260 / 280 measured with the Nanodrop 8000. 260 / 230 ratios between 2.0-2.2 are indicative of DNA samples free from organic contaminants. 260 / 280 ratios below 2.0 are indicative of a sample with minimal RNA. Samples treated by Buffers 1, 2, and 3 were identified as meeting the criteria stated before for both purity ratios, indicating the non-ionic detergents in the buffers were least impactful on the purity of the samples. Downstream testing with agarose gel electrophoresis identified more damage to the.

[0078] FIG.1 and FIG.2 demonstrate that a variety of non-ionic surfactants are effective at isolating pDNA from E. Coli.. Example 4 Effect of Polymyxin on Endotoxin removal

[0079] Studies were conducted to assess the effect of polymyxin on endotoxin removal. A buffer solution was prepared that consisted of 0.75 M NaCl, 0.05 M MOPS, and 10% Tergitol 15-S-9. Polymyxin-B sulfate was supplemented at 5µg / mL or omitted for comparison. E.coli strain TOP10 transformed with plasmid pSEAP2 was grown overnight for approximately 14- 16 hours in LB broth shaking overnight at 225 rpm in at 37 °C incubator.1 mL of the E.coli culture was added to 30 individual wells of a 96-well KingFisher™ Deep well plate. This plate was processed on the KingFisher™ Apex system.45 uL of Buffer 2 was used to treat samples for endotoxin, and samples quantified with the Nanodrop 8000. Endotoxin quantification was done using the Charles River Laboratories Endosafe Nexgen™ PTS system on five representative samples. Samples treated with buffer 2 containing polymyxin B sulfate had theAttorney Docket No. TP387448WO1 lowest Eu / ug concentration compared to samples treated by buffer 2 without polymyxin B sulfate, indicating that polymyxin B sulfate is necessary for endotoxin extraction in the mini- prep workflow (FIG.3). Example 5 Effect of polymyxin levels on endotoxin reduction

[0080] Studies were conducted on the effect of polymyxin B sulfate levels on endotoxin reduction in pDNA isolates in E. coli cell lysates. A buffer solution was prepared that consisted of 0.75 M NaCl, 0.05 M MOPS, and 10% Tergitol 15-S-9. Using this buffer as a base, polymyxin-B sulfate was supplemented at concentrations of 2.75 mg / mL, 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, and 10 mg / mL. E.coli strain TOP10 transformed with plasmid pSEAP2 was grown overnight for approximately 14-16 hours in LB broth shaking overnight at 225 rpm in at 37 °C incubator. 1 mL of the E.coli culture was added to 40 individual wells of a 96-well KingFisher™ Deep well plate. This plate was processed on the KingFisher™ Apex system. 45 uL of Buffer 2 at the varying concentrations was used to treat samples for endotoxin, and samples quantified with the Nanodrop 8000. Endotoxin quantification was done using the Charles River Laboratories Endosafe Nexgen PTS system on nine representative samples. Samples treated with Buffer 2 supplemented by 5 mg / mL, 6 mg / mL, 7 mg / mL, 8 mg / mL, and 10 mg / mL demonstrated 98-99% reduction in endotoxin concentration compared to untreated control samples and samples treated with buffer 2 supplemented by 2.75 mg / mL of polymyxin B sulfate (FIG.4).

[0081] The compositions, methods, and kits of the current teachings have been described broadly and generically herein. Each of the narrower species and sub-generic groupings falling within the generic disclosure also form part of the current teachings. This includes the generic description of the current teachings with a proviso or negative limitation removing any subject matter from the genus, regardless of whether or not the excised material is specifically recited herein.

[0082] Although the disclosed teachings have been described with reference to various applications, methods, and compositions, it will be appreciated that various changes and modifications can be made without departing from the teachings herein. The foregoingAttorney Docket No. TP387448WO1 examples are provided to better illustrate the present teachings and are not intended to limit the scope of the teachings herein. Certain aspects of the present teachings can be further understood in light of the following claims.

Claims

Attorney Docket No. TP387448WO1 WHAT IS CLAIMED IS:

1. A composition comprising, in aqueous solution: (a) a non-ionic detergent; and (b) a polymyxin.

2. The composition of claim 1, further comprising a buffering agent.

3. The composition of claim 1 or 2, further comprising an alkali or alkaline earth metal salt.

4. The composition of claim 2, wherein the buffering agent is present at a concentration of 10mM to 100mM.

5. The composition of claim 3 or 4, wherein the alkali or alkaline earth metal salt is present at a concentration of 0.1M to 1.5M.

6. The composition of any of the preceding claims, wherein the non-ionic detergent is present at a concentration of 0.05% to about 25%.

7. The composition of any of the preceding claims, wherein the polymyxin is present at a concentration of 0.5mg / mL to 15mg / mL 8. The composition of any of the preceding claims, wherein the alkali or alkaline earth metal salt is selected from the group consisting of LiCl, NaCl, KCl, MgCl2, BaCl2, and CaCl2, and corresponding acetates, or any combination thereof.

9. The composition of any of the preceding claims, wherein the non-ionic detergent is selected from the group consisting of Ecosurf™ EH-9 (ethylene oxide-propylene oxide copolymer mono(2-ethylhexyl) ether), Ecosurf™ SA-4 (Seed oil alcohol ethoxylate 4 EO), Ecosurf™ SA-9 (seed oil alcohol ethoxylate 9 EO or polyethylenglykolether), Ecosurf™ EH-6 (2-Ethyl hexanol EO-PO nonionic surfactant), Ecosurf™ EH-3 (2-Ethyl hexanol EO-PO nonionic surfactant), SaponinAttorney Docket No. TP387448WO1 (triterpene glycosides), Ecosurf™, Tween® 80 (Polyoxyethylenesorbitan monooleate), Tween® 85 (Polyoxyethylenesorbitan Trioleate), Tween® 40 (Polyoxyethylenesorbitan monopalmitate), Tween® 20 (Polyoxyethylenesorbitan monolaurate), Tween® 60 (Polyoxyethylene sorbitan monostearate), Tween® 65 (Polyoxyethylenesorbitan Tristearate), Tergitol 15-S-7 (Alcohols, C12- 14-secondary, ethoxylated), Tergitol 15-S-9 (sek-alkoxypolyethylenglycol or Alcohols, C11-15- secondary, ethoxylated), Tergitol 15-S-12, Tergitol 15-S-30 (Secondary alcohol ethoxylate 31EO), Tergitol 15-S-40 (Secondary alcohol ethoxylate 41 EO), Tergitol NP-11, Tergitol NP-13, Tergitol NP-50, Tergitol NP-30 (nonyl phenol polyglycol ether 30 ehthoxylate), Tergitol NP-15, Tergitol NP- 40, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S-20, Tergitol NP-70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S-5, Pluronic F-127 (2-[2-(2- hydroxyethoxy)propoxy]ethanol), Synperonic® F 108 (Poly(ethylene glycol)-block-poly(propylene glycol)-block-poly(ethylene glycol), Synperonic® PE P105 (ethylene oxide / propylene oxide block copolymer), SA-7, Poloxamer 188 (Polyethylene-Polypropylene Glycol) , Triton X-45 (4-(1,1,3,3- Tetramethylbutyl)phenyl-polyethylene glycol), Triton X-100 (t-Octylphenoxypolyethoxyethanol), Triton X-114 (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol), Triton X-102 (Octyl Phenol Ethoxylate), Triton X-165 (Polyethylene glycol tert-octylphenyl ether), Triton X-305 (t- Octylphenoxypolyethoxyethanol), Triton X-705 (2-[4-(2,4,4-trimethylpentan-2- yl)phenoxy]ethanol), Triton™ X-405 (4-tert-octylphenol monoethoxylate), Triton™ X-405, reduced, Triton™ X-100 reduced, Triton™ N-101 (Polyethylene glycol nonaphenyl ether Alkylphenol ethoxylates), reduced, Triton™ CG-110 ((3R,4S,5S,6R)-2-decoxy-6-(hydroxymethyl)oxane-3,4,5- triol), Brij® 35 (Polyoxyethylene lauryl ether), Brij® 58 (Polyethylene glycol hexadecyl ether), Brij® L23 (Polyoxyethylene (23) lauryl ether), Brij® S10 (Polyethylene glycol octadecyl ether), BRIJ® O20 (Polyoxyethylene (20) oleyl ether), Brij® S 100 (Polyoxyethylene (100) stearyl ether), Brij® O10 (Polyoxyethylene (10) oleyl ether), Brij® S20 (docosaethylene glycol mono octadecyl ether), Brij® C10 (Polyoxyethylene (10) cetyl ether), Brij® L4 (Polyoxyethylene (4) lauryl ether), Brij® 93 (Polyoxyethylene (2) oleyl ether), SP Brij® S2 MBAL (diethylene glycol octadecyl ether), Digitonin, MERPOL® A, MERPOL® HCS, MERPOL® SH, MERPOL® SE, Elugent, Octyl β-D- glucopyranoside, n-Dodecyl β-D-maltoside, Decyl β-D-maltopyranoside, n-Octyl β-D-maltoside, Decyl β-D-glucopyranoside, Octyl α-D-glucopyranoside, Hexyl β-D-glucopyranoside, Nonyl β-D- maltoside, IGEPAL® CA-630 (2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethan-1-ol), IGEPAL® CO-520 (Polyoxyethylene (5) nonylphenylether, branched), IGEPAL® CO-630 (Polyoxyethylene (9)Attorney Docket No. TP387448WO1 nonylphenylether), IGEPAL® CA-720 (Polyoxyethylene (12) isooctylphenyl ether), IGEPAL® CO- 890 (Polyoxyethylene (40) nonylphenyl ether, branched), Octyl-beta-Glucoside, Octylthio Glucoside, cocoamide monoethanolamine (Cocamide MEA), cocamide diethanolamine (Cocamide DEA), or any combination thereof.

9. The composition of any of the preceding claims, wherein the polymyxin is selected from the group consisting of polymyxin-B and polymyxin-E.

10. The composition of any of claims 2-9, wherein the buffering agent is selected from the group consisting of N-2-acetamido-2-aminoethanesulfonic acid (ACES), N-2-acetamido-2- iminodiacetic acid (ADA), 3-1,1-dimethyl-2-hydroxyethylamino-2-hydroxy propanesulfonic acid (AMPSO), N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES), 4-cyclohexylamino-1-butane sulfonic acid (CABS), 3-cyclohexylamino-1-propane sulfonic acid (CAPS), 3-cyclohexylamino-2- hydroxy-1-propane sulfonic acid (CAPSO), 2-N-cyclohexylaminoethanesulfonic acid (CHES), 3- N,N-bis-2-hydroxyethylamino-2-hydroxypropanesulfonic acid (DIPSO), N-2- hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS or HEPPS), N-2-hydroxyethylpiperazine- N-4-butanesulfonic acid (HEPBS), 4-N-morpholinobutanesulfonic acid (MOBS), 3-N-morpholino- 2-hydroxypropanesulfonic acid (MOPSO), Bis-Tris, hydroxyethylpiperazine ethane sulfonic acid (HEPES), morpholinoethanesulfonic acid (MES), 3-(N-morpholino)propanesulfonic acid (MOPS), [tris(hydroxymethyl)methylamino] propanesulfonic acid (TAPS), N-trishydroxymethyl-methyl-4- aminobutanesulfonic acid (TABS), N-trishydroxymethyl-methyl-3-aminopropanesulfonic acid (TAPS), 3-N-trishydroxymethyl-methylamino-2-hydroxypropanesulfonic acid (TAPSO), N- trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES), N-(2-acetamido)iminodiacetic acid (ADA), Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), Tris-Hydrochloride (Tris-HCl), N- trishydroxymethylmethylglycine (TRICINE), or any combination thereof.

11. A composition comprising, in aqueous solution: (a) an alkali or alkaline earth metal salt at concentrations of about 0.1 M to about 1.5 M, wherein the alkali metal salt is selected from the group consisting of LiCl, NaCl, and KCl, or any combination thereof,Attorney Docket No. TP387448WO1 (b) a non-ionic detergent in amounts of about 0.051% to about 25%, wherein the non-ionic detergent is selected from the group consisting of Ecosurf™ EH-9, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™, Tween® 80, Tween® 85, Tween® 40, Tween® 20, Tween® 60, Tween® 65, Tergitol 15-S-7, Tergitol 15-S-9, Tergitol 15-S- 12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, Tergitol NP-50, Tergitol NP- 30, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S-20, Tergitol NP-70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S-5, Pluronic F-127, Synperonic® F 108, Synperonic® PE P105, SA-7, Poloxamer 188, , Triton X-45, Triton X-100, Triton X-114, Triton X-102, Triton X-165, Triton X-305, Triton X-705, Triton™ X-405, Triton™ X- 405, reduced, Triton™ X-100 reduced, Triton™ N-101, reduced, Triton™ CG-110, Brij® 35, Brij® 58, Brij® L23, Brij® S10, BRIJ® O20, Brij® S 100, Brij® O10, Brij® S20, Brij® C10, Brij® L4, Brij® 93, SP Brij® S2 MBAL, Digitonin, MERPOL® A, MERPOL® HCS, MERPOL® SH, MERPOL® SE, Elugent, Octyl β-D-glucopyranoside, n-Dodecyl β-D-maltoside, Decyl β-D- maltopyranoside, n-Octyl β-D-maltoside, Decyl β-D-glucopyranoside, Octyl α-D-glucopyranoside, Hexyl β-D-glucopyranoside, Nonyl β-D-maltoside, IGEPAL® CA-630, IGEPAL® CO-520, IGEPAL® CO-630, IGEPAL® CA-720, IGEPAL® CO-890, Octyl-beta-Glucoside, Octylthio Glucoside, cocoamide monoethanolamine (Cocamide MEA), cocamide diethanolamine (Cocamide DEA), or any combination thereof, (c) a polymyxin at concentrations of about 0.5 mg / mL to about 15 mg / mL, wherein the polymyxin is selected from the group consisting of polymyxin-B and polymyxin-E; and (d) a buffering agent at a concentration of about 10 mM to about 100 mM, wherein the buffering agent is selected from the group consisting of N-2-acetamido-2-aminoethanesulfonic acid (ACES), N-2-acetamido-2-iminodiacetic acid (ADA), 3-1,1-dimethyl-2-hydroxyethylamino-2- hydroxy propanesulfonic acid (AMPSO), N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES), 4-cyclohexylamino-1-butane sulfonic acid (CABS), 3-cyclohexylamino-1-propane sulfonic acid (CAPS), 3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO), 2-N- cyclohexylaminoethanesulfonic acid (CHES), 3-N,N-bis-2-hydroxyethylamino-2- hydroxypropanesulfonic acid (DIPSO), N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS or HEPPS), N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS), 4-N- morpholinobutanesulfonic acid (MOBS), 3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris),Attorney Docket No. TP387448WO1 hydroxyethylpiperazine ethane sulfonic acid (HEPES), morpholinoethanesulfonic acid (MES), 3-(N- morpholino)propanesulfonic acid (MOPS), [tris(hydroxymethyl)methylamino] propanesulfonic acid (TAPS), N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS), N-trishydroxymethyl- methyl-3-aminopropanesulfonic acid (TAPS), 3-N-trishydroxymethyl-methylamino-2- hydroxypropanesulfonic acid (TAPSO), N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES), N-(2-acetamido)iminodiacetic acid (ADA), Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), N- trishydroxymethylmethylglycine (TRICINE), or any combination thereof.

12. The composition of any of the preceding claims, further comprising a lysis buffer, wherein the lysis buffer comprises a denaturing agent.

13. The composition of any of the preceding claims, further comprising a re-suspension buffer, wherein the resuspension buffer comprises an RNase.

14. The composition of any of the preceding claims, further comprising a precipitation buffer, wherein the precipitation buffer comprises an acid.

15. A kit comprising a composition of any of claims 1-11, and optionally one or more of a lysis buffer comprising a denaturing agent; a re-suspension buffer comprising an RNase; and a precipitation buffer comprising an alkali metal salt.

16. A method of preparing endotoxin-free plasmid DNA (pDNA) from a sample comprising a microorganism, wherein the microorganism comprises the pDNA, the method comprising: (a) providing a sample comprising the microorganism; (b) lysing the microorganism in the presence of a buffer comprising a non-ionic surfactant, and polymyxin to produce a mixture comprising cellular debris and pDNA; (c) isolating the pDNA from the mixture.

17. The method of claim 16, wherein the buffer further comprises:Attorney Docket No. TP387448WO1 a buffering agent; and an alkaline or alkali metal salt, or combination thereof.

18. The method of claim 16 or 17, wherein the lysing step comprises chemical lysis with an alkali metal salt selected from NaOH, KOH, or a combination thereof, or with a guanidium salt.

19. The method of claim 18, wherein the chemical lysis comprises lysing with a NaOH or KOH or combination thereof, and wherein the method further comprises adding a neutralizing buffer to precipitate the cellular debris from the mixture.

20. The method of claim 16, wherein the isolating step comprises, prior to step (b): contacting the sample with first a solid phase, wherein the microorganism is a gram-negative microorganism that binds to the first solid phase.

21. The method of claim 20, wherein the cellular debris selectively binds to the first solid phase, and wherein the pDNA does not bind to the first solid phase.

22. The method of claim 21, further comprising removing the first solid phase with the cellular debris bound thereto from the mixture, to produce a pDNA-enriched mixture.

23. The method of claim 22, further comprising: adding a second solid phase and an endotoxin removal buffer to the pDNA enriched mixture, wherein in the presence of the endotoxin removal buffer, the pDNA selectively binds to the second solid phase; and eluting the pDNA from the second solid phase.

24. The method of claim 23, further comprising washing the pDNA-bound second solid phase prior to eluting the pDNA from the second solid phase.Attorney Docket No. TP387448WO1 25. The method of claim 23, wherein the washing comprises at least one step of contacting the pDNA-bound second solid phase with an ammonium sulphate solution, water, or both.

26. A method for preparing plasmid DNA (pDNA) from a biological sample comprising a microorganism, wherein the microorganism comprises the pDNA, comprising: (a) contacting the biological sample with a first solid phase under conditions which selectively bind the microorganism; (b) incubating the microorganism-bound first solid phase with a lysis / resuspension buffer and an RNase at an incubation temperature and for a time to produce a lysate; (c) incubating the lysate with a precipitation buffer at an incubation temperature and for a time to produce a first mixture comprising cellular debris and pDNA, wherein the cellular debris selectively binds to the first solid phase; (d) removing the cellular debris-bound first solid phase from the second mixture to produce a pDNA-enriched solution; (e) adding a second solid phase and an endotoxin removal buffer to the pDNA- enriched solution to produce a second mixture, wherein the pDNA selectively binds to the second solid phase in the second mixture; and (f) eluting the pDNA from the pDNA bound second solid phase.

27. The method of claim 26, wherein after step (a), the microorganism-bound first solid phase is removed from the biological sample and resuspended in the lysis buffer of step (b).

28. The method of claim 26, further comprising: (c) washing the pDNA bound second solid phase with ammonium salts; (d) washing the pDNA bound second solid phase with water; (e) contacting the second solid phase from step (d) with an elution buffer for a time and at a temperature releasing the pDNA from the second solid phase into the elution buffer; and wherein the pDNA from the endotoxin free lysate is compatible with in situ polymerase or reverse transcription reactions.Attorney Docket No. TP387448WO1 29. The method of claim 28, wherein the pDNA binds the second solid phase at a pH of less than about 6.0 and is released from the second solid phase at a pH of greater than about 8.

0.

30. The method of any of claims 26-29, wherein the pH of the lysis buffer is between about 11.8 and about 14.

0.

31. The method of any of claims 26-29, wherein the pH of the precipitation buffer is between about 3.5 and about 5.

5.

32. The method of any of claims 26-29, wherein the pH of the elution buffer is between about 8.0 and about 9.

5.

33. The method of any of claims 26-32, wherein the first and second solid phase is controlled pore glass, a polysaccharide, a ceramic material, a porous plastic material, a plastic material, polystyrene bead, bead, particle, tube, well, probe, dipstick, pipette tip, slide, fiber, membrane, paper, cellulose, agarose, glass, or plastic, and wherein the pDNA is released at a pH of between about 8.0 and about 10.

0. 34 The method of any of claims 26-32wherein the first and second solid phase is magnetic beads.

35. The method of any of claims 26-32, wherein the first and second solid phase are paramagnetic beads.

36. The method of any of claims 26-32, wherein the first and second solid phase comprise a plurality of ionizable groups selected from the group consisting of polyhydroxylated amines, histidine, polyamines, imidazole, and poly-histidine.

37. The method of any of claims 26-32, wherein the first and second solid phase comprise a plurality of ionizable groups selected from the group consisting of N- trishydroxymethylmethylglycine (TRICINE), trishydroxymethylaminomethane (Tris),Attorney Docket No. TP387448WO1 polyhydroxylated imidazoles, bis-2-hydroxyethyliminotrishydroxymethylmethane (Bis-Tris), 1,3-bistrishydroxymethylmethylaminopropane (Bis-TrisPropane), N- trishydroxymethylmethylglycine (TRICINE), trishydroxymethylaminomethane (Tris), polyhydroxylated imidazoles, triethanolamine dimers and polymers, heterocyclic nitrogen-containing aromatic or aliphatic compounds and may be monomers, oligomers or polymers, such as morpholine- , pyrrole-, pyrrolidine-, pyridine-, pyridinol-, pyridone-, pyrroline-, pyrazole-, pyridazine-, pyrazine- , piperidone-, piperidine-, or piperazine-containing compounds.

38. The method of any of claims 26 to 37, wherein the first and second solid phase has a pKa between about 4.5 and about 8.

5.

39. A kit comprising a buffer for isolating pDNA from a biological sample, wherein the said buffer comprises: (a) an alkali or alkaline earth metal salt at concentrations of about 0.1 M to about 1.5 M, (b) a non-ionic detergent in amounts of about 0.05% to about 25%, (c) a polymyxin at concentrations of about 0.5 mg / mL to about 15 mg / mL; and (d) a buffering agent at a concentration of about 10 mM to about100 mM.

40. The kit according to claim 39, further comprising: (a) a lysis buffer, comprising a denaturing agent, (b) a re-suspension buffer comprising a RNase, (c) a precipitation buffer comprising an alkali metal salt, and (d) an elution buffer comprising a buffering agent and an alkali metal hydroxide.

41. The kit of claim 40, wherein the pH of the lysis buffer is between about 11.8 and about 14.

0.

42. The kit of claim 40, wherein the pH of the resuspension buffer is between about 8.0 and about 10.0.Attorney Docket No. TP387448WO1 43. The kit of claim 40, wherein the pH of the precipitation buffer is between about 3.5 and about 5.

5.

44. The kit of claim 40, wherein the pH of the elution buffer is between about 8.0 and about 9.

5.

45. A composition for endotoxin removal comprising, in an aqueous solution: (a) an alkali or alkaline earth metal salt at concentrations of about 0.1 M to about 1.5 M, (b) a non-ionic detergent in amounts of about 0.05% to about 25%, (c) a polymyxin at concentrations of about 0.5 mg / mL to about 15 mg / mL; and (d). a buffering agent at a concentration of about 10 mM to about100 mM.

46. A composition for endotoxin removal comprising, in an aqueous solution: (a) an alkali or alkaline earth metal salt at concentrations of about 0.1 M to about 1.5 M, wherein the alkali metal salt is selected from the group consisting of LiCl, NaCl, and KCl, or any combination thereof, (b) a non-ionic detergent in amounts of about 0.05% to about 25%, wherein the non-ionic detergent is selected from the group consisting of Ecosurf™ EH-9, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™, Tween® 80, Tween® 85, Tween® 40, Tween® 20, Tween® 60, Tween® 65, Tergitol 15-S-7, Tergitol 15-S-9, Tergitol 15-S- 12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, Tergitol NP-50, Tergitol NP- 30, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S-20, Tergitol NP-70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S-5, Pluronic F-127, Synperonic® F 108, Synperonic® PE P105, SA-7, Poloxamer 188, , Triton X-45, Triton X-100, Triton X-114, Triton X-102, Triton X-165, Triton X-305, Triton X-705, Triton™ X-405, Triton™ X- 405, reduced, Triton™ X-100 reduced, Triton™ N-101, reduced, Triton™ CG-110, Brij® 35, Brij® 58, Brij® L23, Brij® S10, BRIJ® O20, Brij® S 100, Brij® O10, Brij® S20, Brij® C10, Brij® L4, Brij® 93, SP Brij® S2 MBAL, Digitonin, MERPOL® A, MERPOL® HCS, MERPOL® SH, MERPOL® SE, Elugent, Octyl β-D-glucopyranoside, n-Dodecyl β-D-maltoside, Decyl β-D- maltopyranoside, n-Octyl β-D-maltoside, Decyl β-D-glucopyranoside, Octyl α-D-glucopyranoside,Attorney Docket No. TP387448WO1 Hexyl β-D-glucopyranoside, Nonyl β-D-maltoside, IGEPAL® CA-630, IGEPAL® CO-520, IGEPAL® CO-630, IGEPAL® CA-720, IGEPAL® CO-890, Octyl-beta-Glucoside, Octylthio Glucoside, cocoamide monoethanolamine (Cocamide MEA), cocamide diethanolamine (Cocamide DEA), or any combination thereof, (c) a polymyxin at concentrations of about 0.5 mg / mL to about 15 mg / mL, wherein the polymyxin is selected from the group consisting of polymyxin-B and polymyxin-E; and (d) a buffering agent at a concentration of about 10 mM to about 100 mM, wherein the buffering agent is selected from the group consisting of N-2-acetamido-2-aminoethanesulfonic acid (ACES), N-2-acetamido-2-iminodiacetic acid (ADA), 3-1,1-dimethyl-2-hydroxyethylamino-2- hydroxy propanesulfonic acid (AMPSO), N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES), 4-cyclohexylamino-1-butane sulfonic acid (CABS), 3-cyclohexylamino-1-propane sulfonic acid (CAPS), 3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO), 2-N- cyclohexylaminoethanesulfonic acid (CHES), 3-N,N-bis-2-hydroxyethylamino-2- hydroxypropanesulfonic acid (DIPSO), N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (EPPS or HEPPS), N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS), 4-N- morpholinobutanesulfonic acid (MOBS), 3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris), hydroxyethylpiperazine ethane sulfonic acid (HEPES), morpholinoethanesulfonic acid (MES), 3-(N- morpholino)propanesulfonic acid (MOPS), [tris(hydroxymethyl)methylamino] propanesulfonic acid (TAPS), N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS), N-trishydroxymethyl- methyl-3-aminopropanesulfonic acid (TAPS), 3-N-trishydroxymethyl-methylamino-2- hydroxypropanesulfonic acid (TAPSO), N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES), N-(2-acetamido)iminodiacetic acid (ADA), Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), N- trishydroxymethylmethylglycine (TRICINE), or any combination thereof.

47. Use of a kit for isolating pDNA from a biological sample, the kit comprising a buffer, wherein the said buffer comprises: (a) an alkali or alkaline earth metal salt at concentrations of about 0.1 M to about 1.5 M, (b) a non-ionic detergent in amounts of about 0.05% to about 25%,Attorney Docket No. TP387448WO1 (c) a polymyxin at concentrations of about 0.5 mg / mL to about 15 mg / mL; and (d) a buffering agent at a concentration of about 10 mM to about100 mM.

48. Use according to claim 47, characterized in that, the kit further comprising: (a) a lysis buffer, comprising a denaturing agent, (b) a re-suspension buffer comprising a RNase, (c) a precipitation buffer comprising an alkali metal salt, and (d) an elution buffer comprising a buffering agent and an alkali metal hydroxide.

49. Use according to claim 48, characterized in that, pH of the lysis buffer is between about 11.8 and about 14.

0.

50. Use according to claim 48, characterized in that, pH of the resuspension buffer is between about 8.0 and about 10.

0.

51. Use according to claim 48, characterized in that, pH of the precipitation buffer is between about 3.5 and about 5.

5.

52. Use according to claim 48, characterized in that, pH of the elution buffer is between about 8.0 and about 9.

5.

53. Use of a composition for endotoxin removal characterized in that, the composition comprises, in an aqueous solution: (a) an alkali or alkaline earth metal salt at concentrations of about 0.1 M to about 1.5 M, (b) a non-ionic detergent in amounts of about 0.05% to about 25%, (c) a polymyxin at concentrations of about 0.5 mg / mL to about 15 mg / mL; and (d). a buffering agent at a concentration of about 10 mM to about100 mM.

54. Use of a composition for endotoxin removal characterized in that, the composition comprises, in an aqueous solution:Attorney Docket No. TP387448WO1 (a) an alkali or alkaline earth metal salt at concentrations of about 0.1 M to about 1.5 M, wherein the alkali metal salt is selected from the group consisting of LiCl, NaCl, and KCl, or any combination thereof, (b) a non-ionic detergent in amounts of about 0.05% to about 25%, wherein the non-ionic detergent is selected from the group consisting of Ecosurf™ EH-9, Ecosurf™ SA-4, Ecosurf™ SA-9, Ecosurf™ EH-6, Ecosurf™ EH-3, Saponin, Ecosurf™, Tween® 80, Tween® 85, Tween® 40, Tween® 20, Tween® 60, Tween® 65, Tergitol 15-S-7, Tergitol 15-S-9, Tergitol 15-S- 12, Tergitol 15-S-30, Tergitol 15-S-40, Tergitol NP-11, Tergitol NP-13, Tergitol NP-50, Tergitol NP- 30, Tergitol NP-15, Tergitol NP-40, Tergitol NP-8, Tergitol 26-7, Tergitol 15-S-20, Tergitol NP-70, Tergitol NP-40, Tergitol TMN6, Tergitol TMN-3, Tergitol 15-S-15, Tergitol 15-S-5, Pluronic F-127, Synperonic® F 108, Synperonic® PE P105, SA-7, Poloxamer 188, , Triton X-45, Triton X-100, Triton X-114, Triton X-102, Triton X-165, Triton X-305, Triton X-705, Triton™ X-405, Triton™ X- 405, reduced, Triton™ X-100 reduced, Triton™ N-101, reduced, Triton™ CG-110, Brij® 35, Brij® 58, Brij® L23, Brij® S10, BRIJ® O20, Brij® S 100, Brij® O10, Brij® S20, Brij® C10, Brij® L4, Brij® 93, SP Brij® S2 MBAL, Digitonin, MERPOL® A, MERPOL® HCS, MERPOL® SH, MERPOL® SE, Elugent, Octyl β-D-glucopyranoside, n-Dodecyl β-D-maltoside, Decyl β-D- maltopyranoside, n-Octyl β-D-maltoside, Decyl β-D-glucopyranoside, Octyl α-D-glucopyranoside, Hexyl β-D-glucopyranoside, Nonyl β-D-maltoside, IGEPAL® CA-630, IGEPAL® CO-520, IGEPAL® CO-630, IGEPAL® CA-720, IGEPAL® CO-890, Octyl-beta-Glucoside, Octylthio Glucoside, cocoamide monoethanolamine (Cocamide MEA), cocamide diethanolamine (Cocamide DEA), or any combination thereof, (c) a polymyxin at concentrations of about 0.5 mg / mL to about 15 mg / mL, wherein the polymyxin is selected from the group consisting of polymyxin-B and polymyxin-E; and (d) a buffering agent at a concentration of about 10 mM to about 100 mM, wherein the buffering agent is selected from the group consisting of N-2-acetamido-2-aminoethanesulfonic acid (ACES), N-2-acetamido-2-iminodiacetic acid (ADA), 3-1,1-dimethyl-2-hydroxyethylamino-2- hydroxy propanesulfonic acid (AMPSO), N,N-bis2-hydroxyethyl-2-aminoethanesulfonic acid (BES), 4-cyclohexylamino-1-butane sulfonic acid (CABS), 3-cyclohexylamino-1-propane sulfonic acid (CAPS), 3-cyclohexylamino-2-hydroxy-1-propane sulfonic acid (CAPSO), 2-N- cyclohexylaminoethanesulfonic acid (CHES), 3-N,N-bis-2-hydroxyethylamino-2- hydroxypropanesulfonic acid (DIPSO), N-2-hydroxyethylpiperazine-N-3-propanesulfonic acidAttorney Docket No. TP387448WO1 (EPPS or HEPPS), N-2-hydroxyethylpiperazine-N-4-butanesulfonic acid (HEPBS), 4-N- morpholinobutanesulfonic acid (MOBS), 3-N-morpholino-2-hydroxypropanesulfonic acid (MOPSO), bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Bis-Tris), hydroxyethylpiperazine ethane sulfonic acid (HEPES), morpholinoethanesulfonic acid (MES), 3-(N- morpholino)propanesulfonic acid (MOPS), [tris(hydroxymethyl)methylamino] propanesulfonic acid (TAPS), N-trishydroxymethyl-methyl-4-aminobutanesulfonic acid (TABS), N-trishydroxymethyl- methyl-3-aminopropanesulfonic acid (TAPS), 3-N-trishydroxymethyl-methylamino-2- hydroxypropanesulfonic acid (TAPSO), N-trishydroxymethyl-methyl-2-aminoethanesulfonic acid (TES), N-(2-acetamido)iminodiacetic acid (ADA), Piperazine-N,N′-bis(2-ethanesulfonic acid) (PIPES), tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl), N- trishydroxymethylmethylglycine (TRICINE), or any combination thereof.

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