Conditioning solution for a capillary

Abstract

The presently claimed and described technology is directed to electrophoretic methods for conditioning a capillary using a conditioning buffer including a surface active polymer and a separation reagent including a separation polymer.

Description

[0001] CONDITIONING SOLUTION FOR A CAPILLARY

[0002] RELATED APPLICATIONS

[0003]

[0001] The present patent application claims the priority benefit of U.S. Provisional Patent Application Ser. No. 63 / 517,003, filed August 1, 2023 and U.S. Provisional Patent Application Ser. No. 63 / 632,102, filed April 10, 2024, the content of each is hereby incorporated by reference in its entirety into this disclosure.

[0004] BACKGROUND

[0005]

[0002] Capillary electrophoresis (CE) has been established as a robust technique used by the biopharmaceutical industry to assess the purity and integrity of a diverse set of therapeutics, from monoclonal antibodies to nucleic acids to adeno-associated virus proteins. The degradation of nucleic acids is a vital critical quality attribute (CQA), capillary gel electrophoresis (CGE) offers size-based separation by taking advantage of sieving qualities between different DNA isoform species. The ability to assess the purity and integrity of plasmid preparations has risen in importance as its use as a pre-cursor template to therapeutic mRNA has become accepted and widespread. It is important to characterize the quality of these molecules as they are prone to degradation through storage conditions through temperature handling and sample buffer matrix considerations.

[0006]

[0003] The use of an uncoated capillary greatly improves assay reproducibility by removing coating process variations from the separation. However, electroosmotic flow (EOF) observed with uncoated capillaries, might prevent effective use of such capillaries. Hence, uncoated capillaries used in electrophoretic separations must be conditioned with a conditioning solution before use. Alternatively, separations using uncoated capillaries might be performed with an EOF suppressant added into the separation media. However, both methods have their drawbacks.

[0007]

[0004] The conditioning procedure is important to ensure that the surface of the capillary is fully and uniformly coated. Neutral capillaries use silanes and covalently bonded neutral polymers to shield against the detrimental effects of electroosmotic flow, which creates inherent variation from the manufacturing process. This variation can complicate nucleic acid analysis by altering analyte mobilities to make sizing applications difficult to interpret.

[0008]

[0005] For uncoated capillaries, typical conditioning and regeneration techniques can require significant time and specific conditions, which can prevent usage and / or impact the efficiency of the capillary and instrumentation. For example, conditioning techniques might require the use of reagents with high viscosity, which require more time for the coating and might result in non- uniform coating. Further, conditioning techniques might require conditioning the capillary before each separation, therefore prolonging the analysis time. Further, prior approaches utilize polymers that are subject to polymerization process variation for viscosity. Adding EOF suppressant into the separation media might also suffer from drawbacks — e.g., complex and / or time-consuming preparation required for such separation media and suboptimal separation achieved in such mixed matrices.

[0009]

[0006] Further, other components and characteristics of the separation media might play a role in the effectiveness of the separation — for example, ionic strength of the separation media. However, in conventional methods, increasing ionic strength of the separation media was undesirable, as it results in the increase in Joule heating, poor temperature control of the separation run, and poor and / or unreproducible separations. A need exists for the ability to separate and analyze nucleic acid on an uncoated bare-fused silica capillary.

[0010] SUMMARY

[0011]

[0007] The inventors have addressed the current need for a simple reliable method of shielding against the detrimental effects of electroosmotic flow when using uncoated capillaries.

[0012]

[0008] One aspect of the disclosure relates to an electrophoretic method comprising: conditioning an uncoated capillary to produce a conditioned capillary, wherein the conditioning comprises rinsing the uncoated capillary at a temperature of above about 20°C with a conditioning buffer, wherein the conditioning buffer comprises a surface active polymer, and; performing a first separation, wherein the first separation comprises: loading a separation reagent comprising a separation polymer onto the conditioned capillary, loading a first sample comprising at least one nucleic acid onto the conditioned capillary, and applying a voltage to the capillary to effect the separation of the sample; and wherein the surface active polymer and the separation polymer are different.

[0009] In an aspect, the conditioning further comprises rinsing the uncoated capillary with water and / or an acidic solution prior to rinsing with the conditioning buffer

[0013]

[0010] In an aspect, the uncoated capillary is a bare-fused silica capillary.

[0014] [Oi l] In an aspect, rinsing the uncoated capillary occurs at a temperature of about 25°C, alternatively about 26°C, alternatively about 27°C, alternatively about 28°C, alternatively about 29°C, alternatively about 30°C, alternatively about 31°C, alternatively about 32°C, alternatively about 33°C, alternatively about 34°C, alternatively about 35°C, alternatively about 36°C, alternatively about 37°C, alternatively about 38°C, alternatively about 39°C, alternatively about 40°C, alternatively about 41 °C , alternatively about 42°C, alternatively about 43 °C, alternatively about 44°C, alternatively about 45°C, alternatively about 46°C, alternatively about 47°C, alternatively about 48°C, alternatively about 49°C, alternatively about 50°C, alternatively about 51 °C , alternatively about 52°C, alternatively about 53°C, alternatively about 54°C, alternatively about 55°C, alternatively about 56°C, alternatively about 57°C, alternatively about 58°C, alternatively about 59°C, or alternatively about 60°C.

[0015]

[0012] In an aspect, rinsing the uncoated capillary occurs at a temperature of about 39°C, alternatively at a temperature of about 40°C, alternatively at a temperature of about 41 °C, or alternatively at a temperature of about 42°C.

[0016]

[0013] In an aspect, rinsing the uncoated capillary occurs at a temperature of about 25°C, alternatively about 26°C, alternatively about 27°C, alternatively about 28°C, alternatively about 29°C, alternatively about 30°C.

[0017]

[0014] In an aspect, conditioning of the uncoated capillary further comprises voltage-mediated conditioning.

[0018]

[0015] In an aspect, the surface active polymer is a crosslinked polymer, a linear polymer, a slightly branched polymer, an amide, a heterocyclic polymer, polyvinylpyrrolidone, polyethylene oxide, polyethylene glycol, a substituted acrylamide, a methylated acrylamide, a polyvinylalcohol, or a dextran.

[0019]

[0016] In an aspect, the surface active polymer is in a concentration of about 0.005% - about 0.9%, alternatively about 0.005% - about 0.8%, alternatively about 0.005% - about 0.07%, alternatively 0.005% - about 0.06%, alternatively about 0.01% - about 0.9%, alternatively about 0.01% - about 0.8%, alternatively about 0.01% - about 0.07%, alternatively about 0.01% - about 0.06%, alternatively about 0.02% - about 0.9%, alternatively about 0.02% - about 0.8%, alternatively about 0.02% - about 0.07%, alternatively about 0.02% - about 0.06%, alternatively about 0.03% - about 0.9%, alternatively about 0.03% - about 0.8%, alternatively about 0.03% - about 0.07%, alternatively about 0.03% - about 0.06%, alternatively about 0.04% - about 0.9%, alternatively about 0.04% - about 0.8%, alternatively about 0.04% - about 0.07%, alternatively about 0.04% - about 0.06%, alternatively about 0.05% - about 0.9%, alternatively about 0.05% - about 0.8%, alternatively about 0.05% - about 0.07%, alternatively about 0.05% - about 0.06%, alternatively about 0.005% - about 1.5% (w / v), alternatively about 0.01% - about 1.5% (w / v), alternatively about 0.02% - about 1.4% (w / v), alternatively about 0.03% - about 1.3% (w / v), alternatively about 0.4% - about 1.2% (w / v), alternatively about 0.5% - about 1.1% (w / v), alternatively about 0.6% - about 1.0% (w / v), alternatively about 0.7% - about 0.9 % (w / v), alternatively about 0.7% - about 0.8 % (w / v), alternatively about 0.9% - about 0.7% (w / v), alternatively about 0.10% - about 0.6% (w / v), alternatively about 0.11% - about 0.5% (w / v), alternatively about 0.12% - about 0.4% (w / v), alternatively about 0.13% - about 0.3% (w / v), , alternatively about 0.14% - about 0.2 % (w / v), alternatively about 0.005% (w / v), alternatively about 0.007% (w / v), alternatively about 0.009% (w / v), alternatively about 0.01% (w / v), alternatively about 0.15% (w / v), alternatively about 0.02% (w / v), alternatively about 0.025% (w / v), alternatively about 0.03% (w / v), alternatively about 0.035% (w / v), alternatively about 0.04% (w / v), alternatively about 0.045% (w / v), alternatively about 0.05% (w / v), or alternatively about 0.055% (w / v).

[0020]

[0017] In an aspect, the separation is performed at a temperature of between about 20°C and about 30°C, alternatively about 21°C, alternatively about 22°C, alternatively about 23°C, alternatively about 24°C, alternatively about 25°C, alternatively about 26°C, alternatively about 27°C, alternatively about 28°C, or alternatively about 29°C.

[0021]

[0018] In an aspect, the separation polymer is a cellulose derivative or a linear polyacrylamide.

[0022]

[0019] In an aspect, the cellulose derivative is cellulose ether, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, or ethyl cellulose.

[0023]

[0020] In an aspect, the separation reagent has an ionic strength of at least above about 100 mM, alternatively at least above about 200 mM, alternatively at least above about 300 mM, alternatively at least above about 400 mM, alternatively at least above about 500 mM, alternatively at least above about 600 mM, alternatively at least above about 700 mM, alternatively at least above about 800 mM, alternatively at least above about 900 mM, alternatively between about lOOmM - about 900mM.

[0024]

[0021] In an aspect, the separation polymer is in a concentration of about 0.02% - about 3% (w / v), alternatively about 0.03% - about 2.9% (w / v), alternatively about 0.04% - about 2.8% (w / v), alternatively about 0.05% - about 2.7% (w / v), alternatively about 0.06% - about 2.6% (w / v), alternatively about 0.07% - about 2.5% (w / v), alternatively about 0.8% - about 2.4% (w / v), alternatively about 0.9% - about 2.3% (w / v), alternatively about 0.10% - about 2.2% (w / v), alternatively about 0.11% - about 2.1% (w / v), alternatively about 0.12% - about 2.0% (w / v), alternatively about 0.13% - about 1.9% (w / v), alternatively about 0.14% - about 1.8% (w / v), alternatively about 0.15% - about 1.7% (w / v), alternatively about 0.16% - about 1.6% (w / v), alternatively about 0.17% - about 1.5% (w / v), alternatively about 0.18% - about 1.4% (w / v), alternatively about 0.19% - about 1.3% (w / v), or alternatively about 0.2% - about 1.2% (w / v), alternatively about 0.02% (w / v), alternatively about 0.025% (w / v), alternatively about 0.03% (w / v), alternatively about 0.035% (w / v), alternatively about 0.04%, 4% (w / v), alternatively about 0.045% (w / v), alternatively about 0.05% (w / v), alternatively about 0.055% (w / v), alternatively about 0.06% (w / v), alternatively about 0.065% (w / v), alternatively about 0.07% (w / v), alternatively about 0.075% (w / v), alternatively about 0.8% (w / v), alternatively about 0.085% (w / v), alternatively about 0.9% (w / v), alternatively about 0.095% (w / v), alternatively about 0.1% (w / v), alternatively about 0.2% (w / v), alternatively about 0.3% (w / v), alternatively about 0.4% (w / v), alternatively about 0.5% (w / v), alternatively about 0.6% (w / v), alternatively about 0.7% (w / v), alternatively about 0.8% (w / v), or alternatively about 0.9% (w / v).

[0025]

[0022] In an aspect, the separation reagent and / or the first sample further comprise a labeling dye. In an aspect, the labeling dye is added to the at least one nucleic acid prior to loading the first sample.

[0026]

[0023] In an aspect, the first separation further comprises rinsing the conditioned capillary with an acidic solution and / or water prior to loading the separation reagent.

[0027]

[0024] In an aspect, after the first separation the method further comprises at least a second separation, wherein the second separation comprises: rinsing the conditioned capillary with water and / or an acidic solution, loading the separation reagent onto the conditioned capillary, loading a second sample comprising at least one nucleic acid onto the conditioned capillary, and applying a voltage to the capillary to effect the separation of the sample.

[0028]

[0025] In an aspect, the at least one nucleic acid is selected from the group consisting of a plasmid, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), microRNA (miRNA), messenger RNA (mRNA), DNA fragments, and RNA fragments.

[0029]

[0026] In an aspect, the method further comprises detecting the at least one nucleic acid in separated sample.

[0030]

[0027] In an aspect, the method further comprises detecting at least one plasmid isoform. In an aspect, the plasmid isoforms are super coiled, linear, or open circular.

[0031]

[0028] One aspect of the disclosure is a kit for conditioning a capillary comprising a conditioning buffer comprising a surface active polymer, a separation reagent comprising a separation polymer, and instructions for use. In an aspect, the kit further comprises a labeling dye.

[0032]

[0029] In an aspect, the surface active polymer and the separation polymer are different, wherein the surface active polymer is selected from the group consisting of a crosslinked polymer, a linear polymer, a slightly branched polymer, an amide, a heterocyclic polymer, polyvinylpyrrolidone, polyethylene oxide, polyethylene glycol, a substituted acrylamide, a methylated acrylamide, a polyvinylalcohol, and a dextran and the separation polymer is a cellulose derivative or a linear polyacrylamide.

[0033]

[0030] Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments of the disclosure in conjunction with the accompanying figures.

[0034] BRIEF DESCRIPTION OF THE DRAWINGS

[0035]

[0031] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures, wherein:

[0036]

[0032] FIG. 1 is an exemplary workflow according to an aspect of this disclosure.

[0033] FIGs 2A and 2B are exemplary conditioning methods for a 30cm capillary (FIG. 2A) and a 50cm capillary (FIG. 2B).

[0037]

[0034] FIGs 3 A and 3B are exemplary separation methods for a 30cm capillary (FIG. 3 A) and a 50cm capillary (FIG. 3B).

[0038]

[0035] FIGs. 4A and 4B are exemplary shutdown methods for a 30cm capillary (FIG. 4A) and a 50cm capillary (FIG. 4B).

[0039]

[0036] FIGs. 5 A and 5B depict the effect of ionic strength on peak shape on samples separated according to an aspect of this disclosure.

[0040]

[0037] FIGs. 6A-6D depict a DNA separation before and after capillary conditioning done according to an aspect of this disclosure. pUC19 isoforms (FIG. 6A) and GeneRuler Ikb plus DNA ladder (FIG. 6B) was analyzed without and with capillary conditioning. Additionally, ten runs of pUC19 plasmid were analyzed without (FIG. 6C) and with (FIG. 6D) capillary conditioning.

[0041]

[0038] FIG. 7 depicts the separation of a wide size-range of plasmid topoisoforms separated according to an aspect of this disclosure.

[0042]

[0039] FIG. 8 depicts the separation of plasmid isoforms and dsDNA ladder done in accordance with an aspect of this disclosure.

[0043] DETAILED DESCRIPTION

[0044]

[0040] A need exists for the ability to separate and analyze nucleic acid on an uncoated bare- fused silica capillary. In some aspects, this includes the separation and analysis of plasmid topoisoforms. In some aspects, this includes the separation of a wide range of linearized doublestranded DNA. Previous approaches have used normal polarity in uncoated capillaries and leveraged electroosmotic flow (EOF) to help separate plasmid isoforms towards the optical detector. In an embodiment, the disclosed methods use three major components to analyze nucleic acids: i) a bare-fused silica capillary, ii) a conditioning solution including a surface active polymer, and 3) a separation reagent including a dilute solution of a separation polymer. In some aspects, it was discovered that, in contrast to prior approaches, a conditioning solution may suppress EOF (observed with uncoated capillaries) even at a lower concentrations of a surface active polymer. Further, in some aspects, a conditioning solution may do so without the need for conditioning a capillary in between separations (e.g., for > 120 separations). In some aspects, a separation reagent may be different from a surface active polymer (e.g., a separation reagent may not include the surface active polymer). In some aspects, a separation reagent with a lower concentration of a separation polymer may still effectively separate analytes. In some aspects, a separation reagent with a higher an ionic strength (e.g., higher than in prior approaches) may separate analytes in an effective and / or desired way.

[0045]

[0041] Additionally, in some aspects, the disclosed methods provide higher resolution for both plasmid and linear DNA, eliminate the need for polymerization step (e.g., to coat the capillary), thereby improving manufacturability, and allow for the use bare fused silica capillaries for DNA separation, which reduces the cost and improve reproducibility. FIG. 1 depicts an exemplary workflow and instrumental parameters for an embodiment of the disclosed electrophoresis method are illustrated in FIGS. 2A, 2B, 3A, 3B, 4A, and 4C.,

[0046] Definitions

[0047]

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods described herein belong. Any reference to standard methods (e.g., ASTM, TAPPI, AATCC, etc.) refers to the most recent available version of the method at the time of filing of this disclosure unless otherwise indicated.

[0048]

[0043] For any method disclosed herein that includes discrete steps, the steps may be conducted in any feasible order. In addition, as appropriate, any combination of two or more steps may be conducted simultaneously.

[0049]

[0044] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

[0050]

[0045] The words "preferred" and "preferably" refer to embodiments of the invention that may afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0051]

[0046] The term " comprises" and variations thereof do not have a limiting meaning where these terms appear in the description and claims. Such terms will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.

[0052]

[0047] By "consisting of' is meant including, and limited to, whatever follows the phrase "consisting of." Thus, the phrase "consisting of' indicates that the listed elements are required or mandatory, and that no other elements may be present. By "consisting essentially of' is meant including any elements listed after the phrase, and limited to other elements that do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. Thus, the phrase "consisting essentially of' indicates that the listed elements are required or mandatory, but that other elements are optional and may or may not be present depending upon whether or not they materially affect the activity or action of the listed elements.

[0053]

[0048] The singular form "a", "an" and "the" include plural referents unless the context clearly dictates otherwise. These articles refer to one or to more than one (i.e., to at least one). As used herein, the term "or" is generally employed in its usual sense including "and / or" unless the content clearly dictates otherwise. The term "and / or" means any one or more of the items in the list joined by "and / or". As an example, "x and / or y" means any element of the three-element set {(x), (y), (x, y)}. In other words, "x and / or y" means "one or both of x and y". As another example, "x, y, and / or z" means any element of the seven-element set {(x), (y), (z), (x, y), (x, z), (y, z), (x, y, z)}. In other words, "x, y and / or z" means "one or more of x, y and z".

[0054]

[0049] Where ranges are given, endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). Furthermore, unless otherwise indicated or otherwise evident from the context and understanding of one of ordinary skill in the art, values that are expressed as ranges can assume any specific value or subrange within the stated ranges in different embodiments of the disclosure, to the tenth of the unit of the lower limit of the range, unless the context clearly dictates otherwise. Herein, "up to" a number (for example, up to 50) includes the number (for example, 50). The term "in the range" or "within a range" (and similar statements) includes the endpoints of the stated range.

[0055]

[0050] Reference throughout this specification to "one aspect,” "an aspect,” "certain aspects," or "some aspects," etc., means that a particular feature, configuration, composition, or characteristic described in connection with the aspect is included in at least one aspect of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more aspects.

[0056]

[0051] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term "about." As used herein in connection with a measured quantity, the term "about" refers to that variation in the measured quantity as would be expected by the skilled artisan making the measurement and exercising a level of care commensurate with the objective of the measurement and the precision of the measuring equipment used. The term "about" as used in connection with a numerical value throughout the specification and the claims denotes an interval of accuracy, familiar and acceptable to a person skilled in the art. In general, such interval of accuracy is + / -10%. Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0057]

[0052] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0058]

[0053] The term "exemplary" means serving as a non-limiting example, instance, or illustration. As utilized herein, the terms "e.g.," and "for example" set off lists of one or more non-limiting aspects, examples, instances, or illustrations.

[0059]

[0054] As used herein, the term "substantially" refers to the qualitative condition of exhibiting total or near-total extent or degree of a characteristic or property of interest. Biological and chemical phenomena rarely, if ever, go to completion and / or proceed to completeness or achieve or avoid an absolute result. The term "substantially" is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena. For example, "substantially" may refer to being within at least about 20%, alternatively at least about 10%, alternatively at least about 5% of a characteristic or property of interest.

[0060]

[0055] The invention is defined in the claims. However, below is a non-exhaustive listing of non-limiting exemplary aspects. Any one or more of the features of these aspects may be combined with any one or more features of another example, embodiment, or aspect described herein.

[0061] Exemplary Instruments and Methods

[0062]

[0056] Electrophoretic separations may include capillary gel electrophoresis (CGE), capillary zone electrophoresis (CZE), isoelectric focusing (IEF), capillary isoelectric focusing (cIEF), and imaged capillary isoelectric focusing (icIEF).

[0063]

[0057] Commercially available instruments for electrophoretic separations are typically equipped with the following features. A high voltage power supply (e.g., a supply that can apply 0. IkV to 30 kV and handle a current load between 300 and 600 microamperes (pA)), which may also have polarity switching capabilities. A pressure pump (e.g., a pump capable of applying accurate low pressure (0.1- 15 psi) for injections and high pressure (1 to 100 psi) for capillary rinses). A detector — e.g, an ultra-violet (UV) absorbance detector (e.g., at 260 nm for the direct detection of analytes, such as nucleic acids, peptides, or proteins), and / or a laser-induced fluorescence detection (e.g., with an excitation and emission wavelengths compatible with an intercalator dye for low concentration samples).

[0064]

[0058] The capillaries used in capillary gel electrophoresis (CGE) separations may be bare-fused silica (BFS) capillary (e.g., with between 50 to 100 microns inner diameter) with total length, e.g., between about 30 cm and about 50 cm and effective length (length from injection to the detection window) between about 20 cm and about 40 cm. The capillary may be filled with a polymeric gel (the sieving matrix) combined with a separation background electrolyte (e.g., at slightly basic pH). At this pH, nucleic acids are negatively charged due to their phosphate backbone and will inherently have an electrophoretic mobility once an electric field is applied. The analysis of DNA in capillary electrophoresis is anodic, thus requiring the negative polarity at the injection point to migrate the nucleic acids towards the capillary detection window. The use of a BFS capillary with exposed silanol groups require shielding to suppress the electroosmotic flow (EOF), which at negative polarity moves opposite of electrophoretic mobility of the nucleic acids. The surface shielding may be achieved by covalently bonding hydrophilic polymers, such as linear polyacrylamide linked to silanes onto the silica surface. The reduction of EOF may also be achieved by conditioning a capillary or adding EF suppressant into the separation media.

[0065]

[0059] One aspect of the disclosure, an uncoated capillary is treated, or rinsed, with a conditioning buffer at an elevated temperature to produce a conditioned capillary. The uncoated capillary may be, for example, a bare-fused silica capillary. In an aspect, an elevated temperature may be a temperature above room temperature, for example at least above 20°C. In some aspects, the elevated temperature may be above 22°C. In some aspects, the elevated temperature may be above 25°C. In some aspects, the elevated temperature may be above 30°C. In some aspects, the elevated temperature may be above 40°C.

[0066]

[0060] In non-limiting aspects, the elevated temperature may be about 25°C, alternatively about 26°C, alternatively about 27°C, alternatively about 28°C, alternatively about 29°C, alternatively about 30°C, alternatively about 31°C, alternatively about 32°C, alternatively about 33°C, alternatively about 34°C, alternatively about 35°C, alternatively about 36°C, alternatively about 37°C, alternatively about 38°C, alternatively about 39°C, alternatively about 40°C, alternatively about 41°C , alternatively about 42°C, alternatively about 43°C, alternatively about 44°C, alternatively about 45°C, alternatively about 46°C, alternatively about 47°C, alternatively about 48°C, alternatively about 49°C, alternatively about 50°C, alternatively about 51 °C , alternatively about 52°C, alternatively about 53°C, alternatively about 54°C, alternatively about 55°C, alternatively about 56°C, alternatively about 57°C, alternatively about 58°C, alternatively about 59°C, or alternatively about 60°C.

[0067]

[0061] In an aspect, the conditioning buffer includes a surface active polymer. The surface active polymer may be configured to effectively masking surface charges and minimizing capillary wall interactions that would be harmful to nucleic acid separations. Non-limiting examples of the surface active polymer include a crosslinked polymer, a linear polymer, a slightly branched polymer, an amide, a heterocyclic polymer, polyvinylpyrrolidone, polyethylene oxide, polyethylene glycol, a substituted acrylamide, a methylated acrylamide, a polyvinylalcohol, or a dextran. Conditioning at elevated temperatures allows the surface active polymer to interact with the capillary wall and best minimize electroosmotic flow effects.

[0068]

[0062] In some aspects, the surface active polymer is diluted prior to treating, or rinsing, the unconditioned capillary. In non-limiting examples, the surface active polymer may have a concentration of about 0.005% - about 0.9%, alternatively about 0.005% - about 0.8%, alternatively about 0.005% - about 0.07%, alternatively 0.005% - about 0.06%, alternatively about 0.01% - about 0.9%, alternatively about 0.01% - about 0.8%, alternatively about 0.01% - about 0.07%, alternatively about 0.01% - about 0.06%, alternatively about 0.02% - about 0.9%, alternatively about 0.02% - about 0.8%, alternatively about 0.02% - about 0.07%, alternatively about 0.02% - about 0.06%, alternatively about 0.03% - about 0.9%, alternatively about 0.03%

[0069] - about 0.8%, alternatively about 0.03% - about 0.07%, alternatively about 0.03% - about 0.06%, alternatively about 0.04% - about 0.9%, alternatively about 0.04% - about 0.8%, alternatively about 0.04% - about 0.07%, alternatively about 0.04% - about 0.06%, alternatively about 0.05%

[0070] - about 0.9%, alternatively about 0.05% - about 0.8%, alternatively about 0.05% - about 0.07%, alternatively about 0.05% - about 0.06%, alternatively about 0.005% - about 1.5% (w / v), alternatively about 0.01% - about 1.5% (w / v), alternatively about 0.02% - about 1.4% (w / v), alternatively about 0.03% - about 1.3% (w / v), alternatively about 0.4% - about 1.2% (w / v), alternatively about 0.5% - about 1.1% (w / v), alternatively about 0.6% - about 1.0% (w / v), alternatively about 0.7% - about 0.9 % (w / v), alternatively about 0.7% - about 0.8 % (w / v), alternatively about 0.9% - about 0.7% (w / v), alternatively about 0.10% - about 0.6% (w / v), alternatively about 0.11% - about 0.5% (w / v), alternatively about 0.12% - about 0.4% (w / v), alternatively about 0.13% - about 0.3% (w / v), , alternatively about 0.14% - about 0.2 % (w / v), alternatively about 0.005% (w / v), alternatively about 0.007% (w / v), alternatively about 0.009% (w / v), alternatively about 0.01% (w / v), alternatively about 0.15% (w / v), alternatively about 0.02% (w / v), alternatively about 0.025% (w / v), alternatively about 0.03% (w / v), alternatively about 0.035% (w / v), alternatively about 0.04% (w / v), alternatively about 0.045% (w / v), alternatively about 0.05% (w / v), or alternatively about 0.055% (w / v).

[0071]

[0063] In some aspects the use of a low viscosity solution of surface active polymer (e.g., < 0.9%, alternatively < 0.8%, alternatively < 0.07 %, alternatively < 0.06%, alternatively < 0.05%, alternatively < 0.04%, alternatively < 0.03%, alternatively < 0.02%, alternatively < 0.01%, < 0.0095%, alternatively < 0.085%, alternatively < 0.075%, alternatively < 0.065%. alternatively < 0.0055% (w / v)) enables the capillary to be rinsed and coated at temperatures of at least about 25°C, alternatively about 26°C, alternatively about 27°C, alternatively about 28°C, alternatively about 29°C, alternatively about 30°C, alternatively about 31°C, alternatively about 32°C, alternatively about 33°C, alternatively about 34°C, alternatively about 35°C, alternatively about 36°C , alternatively about 371 °C , alternatively about 38°C, alternatively about 39°C, alternatively about 40°C, alternatively about 41 °C , or alternatively about 42°C. This temperature range allows for improved electroosmotic flow (EOF) shielding by optimizing the hydrogen bonding. This, in turn, allows for increased run reproducibility and robust separations.

[0072]

[0064] The surface active polymer may be diluted using a 2-Amino-2-(hydroxymethyl)-l,3- propanediol (Tris) buffer, such as TBE (Tris borate / Ethylenediaminetetraacetic acid (EDTA)) or TAE (Tris acetate / EDTA). The surface active polymer may also be diluted in other compatible electrophoresis buffers such as N,N-bis(2-hydroxyethyl)glycine (bicine), Bis-(2 -Hydroxyethyl) Amino-Tris(Hydroxymethyl) Methane (Bis-Tris), 3 -(cyclohexylamino)- 1 -propane-sulfonic acid (CAPS), EDTA, Ethyleneglycol bis(2-aminoethyl ether)-N,N,N',N' tetraacetic acid (EGTA), 3- (N-morpholino)propanesulfonic acid (MOPS), 2- [4-(2-sulfoethyl)piperazin- 1 -yl] ethanesulfonic acid (PIPES), or N-[Tris(hydroxymethyl)methyl]-3-aminopropanesulfonic acid (TAPS).

[0073]

[0065] In some aspects, the conditioning may further include rinsing the uncoated capillary with water and / or an acidic solution prior to rinsing with the conditioning buffer. In this aspect, rising the capillary with, for example, an acidic solution allows for the protonation of the silica surface silanol groups. This allows for increased interactions for hydrogen bonding with the surface active polymer.

[0074]

[0066] After conditioning, the conditioned capillary may be used in an electrophoretic method, such as an electrophoretic separation. Non-limiting examples of electrophoretic separation include those selected from the group consisting of capillary zone electrophoresis (CZE), CGE, isoelectric focusing (IEF), capillary isoelectric focusing (cIEF), and imaged capillary isoelectric focusing (icIEF). In some aspects, the electrophoretic separation is CGE.

[0075]

[0067] The electrophoretic separations are performed using a separation reagent. The separation reagent may include a separation polymer, which is loaded on to the conditioned capillary. The separation polymer and the surface active polymer are different. In some non-limiting aspects, the separation polymer has a hydrophilicity capable of high peak efficiencies for electrophoretic separations of samples, which include nucleic acid. In some aspects, the separation polymer is also configured to improve peak shape of nucleic acid fragments, especially plasmid isoforms.

[0076]

[0068] In some aspects, the separation polymer may include a cellulose derivative or a linear polyacrylamide. In a non-limiting example, the cellulose derivative is cellulose ether, alternatively hydroxypropyl cellulose, alternatively hydroxypropyl methylcellulose, alternatively hydroxyethyl cellulose, or alternatively ethyl cellulose.

[0077]

[0069] Depending on the separation desired, the separation reagent may have an ionic strength of at least above about 100 mM, alternatively at least above about 200 mM, alternatively at least above about 300 mM, alternatively at least above about 400 mM, alternatively at least above about 500 mM, alternatively at least above about 600 mM, alternatively at least above about 700 mM, alternatively at least above about 800 mM, alternatively at least above about 900 mM, alternatively between about 1 OOmM - about 900mM. In some aspects, the ionic strength can be up to 1 ,3M and still keep adequate peak shape and separation performance. The increase in ionic strength allows the use of dilute polymer concentrations while still maintaining adequate shielding from counter electroosmotic flow. As shown in FIGs. 5A and 5B, the increase of ionic strength improves the peak shape for a linear dsDNA.

[0078]

[0070] The separation polymer may also be diluted prior to loading on to the conditioned capillary. For example the separation polymer may have a concentration of about 0.02% - about 3% (w / v), alternatively about 0.03% - about 2.9% (w / v), alternatively about 0.04% - about 2.8% (w / v), alternatively about 0.05% - about 2.7% (w / v), alternatively about 0.06% - about 2.6% (w / v), alternatively about 0.07% - about 2.5% (w / v), alternatively about 0.8% - about 2.4% (w / v), alternatively about 0.9% - about 2.3% (w / v), alternatively about 0.10% - about 2.2% (w / v), alternatively about 0.11% - about 2.1% (w / v), alternatively about 0.12% - about 2.0% (w / v), alternatively about 0.13% - about 1.9% (w / v), alternatively about 0.14% - about 1.8% (w / v), alternatively about 0.15% - about 1.7% (w / v), alternatively about 0.16% - about 1.6% (w / v), alternatively about 0.17% - about 1.5% (w / v), alternatively about 0.18% - about 1.4% (w / v), alternatively about 0.19% - about 1.3% (w / v), or alternatively about 0.2% - about 1.2% (w / v), alternatively about 0.02% (w / v), alternatively about 0.025% (w / v), alternatively about 0.03% (w / v), alternatively about 0.035% (w / v), alternatively about 0.04%, 4% (w / v), alternatively about 0.045% (w / v), alternatively about 0.05% (w / v), alternatively about 0.055% (w / v), alternatively about 0.06% (w / v), alternatively about 0.065% (w / v), alternatively about 0.07% (w / v), alternatively about 0.075% (w / v), alternatively about 0.8% (w / v), alternatively about 0.085% (w / v), alternatively about 0.9% (w / v), alternatively about 0.095% (w / v), alternatively about 0.1% (w / v), alternatively about 0.2% (w / v), alternatively about 0.3% (w / v), alternatively about 0.4% (w / v), alternatively about 0.5% (w / v), alternatively about 0.6% (w / v), alternatively about 0.7% (w / v), alternatively about 0.8% (w / v), or alternatively about 0.9% (w / v).

[0079]

[0071] In some aspects, the conditioned capillary is rinsed with an acidic solution and / or water prior to loading the separation reagent.

[0080]

[0072] A sample, which includes a nucleic acid, may also be loaded onto the conditioned capillary. This sample may be loaded prior to, with, or after the separation reagent is loaded onto the conditioned capillary. In some aspects, the sample may be pressure driven into the capillary. Pressure injection also called hydrodynamic injection (HDI), requires a small pressure applied at the capillary end opposite to the detection point (capillary inlet) for a short time. The opposite end of the capillary (capillary outlet) is typically immersed in double distilled and deionized water to ensure injection consistency and avoid siphoning effects. In an aspect, conditions for hydrodynamic injection range from 0.3 to 0.5 psi for 3 - 10 seconds.

[0081]

[0073] In some aspects, the separation reagent and / or the sample may include a labeling dye. In some aspects, labeling dye is a fluorescent dye, including, but not limited to a cyanide dye. Non-limiting examples of cyanide dyes include Cy2, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy7, SYBR Gold, SYBR Green I, SYBR Green II, PicoGreen, Thiazole orange, and Oxazole yellow. In some aspects, the labeling dye is added to the nucleic acid prior to loading the sample.

[0082]

[0074] In some aspects, the labeling dye is added to the separation reagent in a general range of about 1:2500 to about 1 :10000 (volume labeling dye : volume of separation reagent). Once an electric field is applied, the nucleic acids migrate towards the detection window and the labeling dye migrates in the opposite direction because it is positively charged. This causes the labeling dye molecules to intercalate with the phosphate groups of the nucleic acid, which then carries it along past the detection point.

[0083]

[0075] Depending on the analysis desired, the sample may include one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more, or ten or more nucleic acid(s). The nucleic acid may be, for example, plasmid, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), microRNA (miRNA), messenger RNA (mRNA), DNA fragments, and RNA fragments. Plasmid DNA can exist in different topological forms including super coiled (or covalently closed circular), linear, or open circular. During plasmid DNA degradation, the super coiled form may become open circular due to single-stranded cleavage. When double-stranded cleave occurs, the plasmid DA will become linear.

[0084]

[0076] In some aspects, a voltage may be applied to the capillary to effect the separation of the sample. In an aspect, the field strength for the separation is from about 200 V / cm - about 500 V / cm. In a 30 cm long capillary, the applied voltage is between about 6,000 V and 15,000 V.

[0085]

[0077] In an aspect, after the separation, the method may further include detecting at least one nucleic acid in separated sample. In another aspect, after separation, the method may further include detecting at least one plasmid isoform. Plasmid DNAs generally exist in 3 major isoforms. In a non-limiting aspect, the plasmid isoforms are super coiled, linear, or open circular (nicked). In some aspects, the separation polymer is selected based on its ability to separate plasmid isoforms, such as dsDNA plasmid isoforms.

[0086]

[0078] In some aspects, the detection utilizes a fluorescence detector. Such as a laser-induced fluorescence (LIF) detector or a lamp-based fluorescence detector. In some aspects, the detection utilizes a UV detector.

[0087]

[0079] The separation, which includes loading a separation reagent onto a conditioned capillary, loading a sample onto a conditioned capillary, and applying a voltage to effect the separation of the sample, may be performed at a temperature of between about 20°C and about 30°C, alternatively about 21°C, alternatively about 22°C, alternatively about 23°C, alternatively about 24°C, alternatively about 25°C, alternatively about 26°C, alternatively about 27°C, alternatively about 28°C, or alternatively about 29°C.

[0088]

[0080] By conditioning the capillary prior to introducing the separation reagent the number of separations required to equilibrate the capillary and have reproducible nucleic acid mobilities is drastically reduced. As shown in FIGs. 6A-6D, the separation efficiency is drastically improved when the capillary is conditioned prior to the separation reagent being introduced. pUC19 isoforms (FIG. 6A) and GeneRuler Ikb plus DNA ladder (FIG. 6B) was analyzed without and with capillary conditioning. Additionally, ten runs of pUC19 plasmid were analyzed without (FIG. 6C) and with (FIG. 6D) capillary conditioning. In each comparison, the resolution and reproducibility is increased.

[0089]

[0081] In some aspects of the disclosed methods, the conditioning is only done prior to a first run in order to prepare the capillary surface for subsequent separations. This allows the conditioned capillary to be used for two or more, alternatively three or more, alternatively four or more, alternatively five or more, alternatively six or more, alternatively seven or more, alternatively eight or more, alternatively nine or more, alternatively ten or more, alternatively twenty or more, alternatively thirty or more, alternatively forty or more, or alternatively fifty or more separations.

[0090]

[0082] In an aspect, the disclosed combination of first coating the capillary with a surface active polymer to suppress EOF followed by a separation in a separation polymer with optimized ionic strength allowed for highly reproducible separations of plasmid and linearized DNA with a long capillary life (e.g., > 120 separations).

[0091]

[0083] When multiple separations are performed, the conditioned capillary is rinsed and the separation polymer replaced between each separation run. Ease of use is provided to the end user by providing ready formulated gel solutions and instrument methods that perform the initial capillary conditioning and subsequent separations.

[0092]

[0084] In an aspect, after a first separation, the method further includes at least a second separation, where the second separation includes rinsing the conditioned capillary with water and / or an acidic solution, loading the separation reagent onto the conditioned capillary, loading a second sample comprising at least one nucleic acid onto the conditioned capillary, and applying a voltage to the capillary to effect the separation of the sample.

[0093]

[0085] Another aspect of the disclosure includes a kit for conditioning a capillary and / or separation. The kit may include a conditioning buffer with a surface active polymer, a separation reagent with a separation polymer, and instructions for use. The surface active polymer and the separation polymer in the kits are different. The surface active polymer may include a crosslinked polymer, a linear polymer, a slightly branched polymer, an amide, a heterocyclic polymer, polyvinylpyrrolidone, polyethylene oxide, polyethylene glycol, a substituted acrylamide, a methylated acrylamide, a polyvinylalcohol, or a dextran. The separation polymer may be a cellulose derivative or a linear polyacrylamide. In various embodiments, the kit may include labeling dyes and / or other reagents required to perform an electrophoretic separation.

[0086] The presently described technology and its advantages will be better understood by reference to the following examples. These examples are provided to describe specific implementations of the present technology. By providing these specific examples, it is not intended limit the scope and spirit of the present technology. It will be understood by those skilled in the art that the full scope of the presently described technology encompasses the subject matter defined by the claims appending this specification, and any alterations, modifications, or equivalents of those claims.

[0094]

[0087] EXAMPLES

[0095]

[0088] Example 1 : Sample Preparation for Exemplary Electrophoretic Nucleic Acid Analysis

[0089] As shown in FIG. 1, a DNA ladder and DNA sample are prepared. The DNA Ladder (5ng / pL) was added to 0. ImM Tris-Borate EDTA (TBE) buffer. The DNA sample was diluted in 0.1 mM TBE or nuclease-free water. The DNA ladder and DNA sample were combined and loaded into a sample vial which was then placed on-instrument at a 10°C sample temperature.

[0096]

[0090] Example 2: Reagent Preparation for Exemplary Electrophoretic Nucleic Acid Analysis

[0091] The reagents for conditioning were prepared and placed into wells of a sample plate. These include water rinse, 0.1M HC1 rinse, and a conditioning buffer including a surface active polymer. The reagents for separation were prepared and placed into their respective vials. These include water rinse, 0.1M HC1 rinse, and a separation reagent including a separation polymer. The separation reagent was mixed with a labeling dye prior to being place into their respective vials.

[0097]

[0092] Example 3 : Sample Analysis for Exemplary Electrophoretic Nucleic Acid Analysis

[0098]

[0093] Capillary electrophoresis analysis was performed using a capillary electrophoresis instruments equipped with a laser induced fluorescence detector (488nm excitation wavelength and a 520nm detection wavelength) and a 30cm or 50cm bare fused capillary. Instrumental parameters for the electrophoresis method are illustrated in FIGS. 2A, 2B, 3A, 3B, 4A, and 4C. For a new capillary, a conditioning method (e.g., FIGs. 2A and 2B) was run at 40°C. For a used capillary, the capillary was rinsed with CE grade water at 50 psi for 5 minutes. The sample was injected into the instrument using pressure, 0.5 psi for 5 seconds, and a separation method (e.g., FIGs. 3A and 3B) was run at 300 V / cm and 25°C. After the completion of sample analysis, a shutdown method (e.g., FIGs. 4A and 4B) was run at 20°C. The sample can be stored long term at temperature between 2°C and 8°C.

[0094] The electropherograms in FIGs. 7 and 8 were generated using the exemplary method. As shown in FIG. 7, the method allows for the separation of plasmids from 2.8 to 18.8kb. The plasmid samples in FIG. 7 were mixed with SYBR Gold prior to analysis. As shown in FIG. 8, the disclosed method enables the DNA separation with a bare fused silica capillary

[0099]

[0095] All features disclosed in the specification, including the claims, abstracts, and drawings, and all the steps in any method or process disclosed, may be combined in any combination, except combinations where at least some of such features and / or steps are mutually exclusive. Each feature disclosed in the specification, including the claims, abstract, and drawings, can be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

[0100]

[0096] It will be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

CLAIMSWhat is Claimed Is:

1. An electrophoretic method comprising: conditioning an uncoated capillary to produce a conditioned capillary, wherein the conditioning comprises rinsing the uncoated capillary at a temperature of above about 20°C with a conditioning buffer, wherein the conditioning buffer comprises a surface active polymer, and; performing a first separation, wherein the first separation comprises: loading a separation reagent comprising a separation polymer onto the conditioned capillary, loading a first sample comprising at least one nucleic acid onto the conditioned capillary, and applying a voltage to the capillary to effect the separation of the sample; and wherein the surface active polymer and the separation polymer are different.

2. The method of claim 1, wherein the conditioning further comprises rinsing the uncoated capillary with water and / or an acidic solution prior to rinsing with the conditioning buffer.

3. The method of any one of the preceding claims, wherein the uncoated capillary is a bare- fused silica capillary.

4. The method of any one of the preceding claims, wherein rinsing the uncoated capillary occurs at a temperature of about 25°C, alternatively about 26°C, alternatively about 27°C, alternatively about 28°C, alternatively about 29°C, alternatively about 30°C, alternatively about 31°C, alternatively about 32°C, alternatively about 33°C, alternatively about 34°C, alternatively about 35°C, alternatively about 36°C, alternatively about 37°C, alternatively about 38°C, alternatively about 39°C, alternatively about 40°C, alternatively about 41 °C , alternatively about 42°C, alternatively about 43°C, alternatively about 44°C, alternatively about 45°C, alternatively about 46°C, alternatively about 47°C, alternatively about 48°C,alternatively about 49°C, alternatively about 50°C, alternatively about 51 °C , alternatively about 52°C, alternatively about 53°C, alternatively about 54°C, alternatively about 55°C, alternatively about 56°C, alternatively about 57°C, alternatively about 58°C, alternatively about 59°C, or alternatively about 60°C.

5. The method of any one of claim 1 to 3, wherein rinsing the uncoated capillary occurs at a temperature of about 39°C, alternatively at a temperature of about 40°C, alternatively at a temperature of about 41 °C, or alternatively at a temperature of about 42°C.

6. The method of any one of the preceding claims, wherein rinsing the uncoated capillary occurs at a temperature of about 25°C, alternatively about 26°C, alternatively about 27°C, alternatively about 28°C, alternatively about 29°C, alternatively about 30°C.

7. The method of any one of the preceding claims, wherein conditioning of the uncoated capillary further comprises voltage-mediated conditioning.

8. The method of any one of the preceding claims, wherein the surface active polymer is a crosslinked polymer, a linear polymer, a slightly branched polymer, an amide, a heterocyclic polymer, polyvinylpyrrolidone, polyethylene oxide, polyethylene glycol, a substituted acrylamide, a methylated acrylamide, a polyvinylalcohol, or a dextran.

9. The method of any one of the preceding claims, wherein the surface active polymer is in a concentration of about 0.005% - about 0.9%, alternatively about 0.005% - about 0.8%, alternatively about 0.005% - about 0.07%, alternatively 0.005% - about 0.06%, alternatively about 0.01% - about 0.9%, alternatively about 0.01% - about 0.8%, alternatively about 0.01% - about 0.07%, alternatively about 0.01% - about 0.06%, alternatively about 0.02% - about 0.9%, alternatively about 0.02% - about 0.8%, alternatively about 0.02% - about 0.07%, alternatively about 0.02% - about 0.06%, alternatively about 0.03% - about 0.9%, alternatively about 0.03% - about 0.8%, alternatively about 0.03% - about 0.07%, alternatively about 0.03% - about 0.06%, alternatively about 0.04% - about 0.9%, alternatively about 0.04% - about 0.8%,alternatively about 0.04% - about 0.07%, alternatively about 0.04% - about 0.06%, alternatively about 0.05% - about 0.9%, alternatively about 0.05% - about 0.8%, alternatively about 0.05% - about 0.07%, alternatively about 0.05% - about 0.06%, alternatively about 0.005% - about 1.5% (w / v), alternatively about 0.01% - about 1.5% (w / v), alternatively about 0.02% - about 1.4% (w / v), alternatively about 0.03% - about 1.3% (w / v), alternatively about 0.4% - about 1.2% (w / v), alternatively about 0.5% - about 1.1% (w / v), alternatively about 0.6% - about 1.0% (w / v), alternatively about 0.7% - about 0.9 % (w / v), alternatively about 0.7% - about 0.8 % (w / v), alternatively about 0.9% - about 0.7% (w / v), alternatively about 0.10% - about 0.6% (w / v), alternatively about 0.11% - about 0.5% (w / v), alternatively about 0.12% - about 0.4% (w / v), alternatively about 0.13% - about 0.3% (w / v), , alternatively about 0.14% - about 0.2 % (w / v), alternatively about 0.005% (w / v), alternatively about 0.007% (w / v), alternatively about 0.009% (w / v), alternatively about 0.01% (w / v), alternatively about 0.15% (w / v), alternatively about 0.02% (w / v), alternatively about 0.025% (w / v), alternatively about 0.03% (w / v), alternatively about 0.035% (w / v), alternatively about 0.04% (w / v), alternatively about 0.045% (w / v), alternatively about 0.05% (w / v), or alternatively about 0.055% (w / v).

10. The method of any one of the preceding claims, wherein the separation is performed at a temperature of between about 20°C and about 30°C, alternatively about 21 °C, alternatively about 22°C, alternatively about 23°C, alternatively about 24°C, alternatively about 25°C, alternatively about 26°C, alternatively about 27°C, alternatively about 28°C, or alternatively about 29°C.

11. The method of any one of the preceding claims, wherein the separation polymer is a cellulose derivative or a linear polyacrylamide.

12. The method of claim 11, wherein the cellulose derivative is cellulose ether, hydroxypropyl cellulose, hydroxypropyl methylcellulose, hydroxyethyl cellulose, or ethyl cellulose.

13. The method of any one of the preceding claims, wherein the separation reagent has an ionic strength of at least above about 100 mM, alternatively at least above about 200 mM, alternatively at least above about 300 mM, alternatively at least above about 400 mM, alternatively at least above about 500 mM, alternatively at least above about 600 mM, alternatively at least above about 700 mM, alternatively at least above about 800 mM, alternatively at least above about 900 mM, alternatively between about 1 OOmM - about900mM.

14. The method of any one of the preceding claims, wherein the separation polymer is in a concentration of about 0.02% - about 3% (w / v), alternatively about 0.03% - about 2.9% (w / v), alternatively about 0.04% - about 2.8% (w / v), alternatively about 0.05% - about 2.7% (w / v), alternatively about 0.06% - about 2.6% (w / v), alternatively about 0.07% - about 2.5% (w / v), alternatively about 0.8% - about 2.4% (w / v), alternatively about 0.9% - about 2.3% (w / v), alternatively about 0.10% - about 2.2% (w / v), alternatively about 0.11% - about 2.1% (w / v), alternatively about 0.12% - about 2.0% (w / v), alternatively about 0.13% - about 1.9% (w / v), alternatively about 0.14% - about 1.8% (w / v), alternatively about 0.15% - about 1.7% (w / v), alternatively about 0.16% - about 1.6% (w / v), alternatively about 0.17% - about 1.5% (w / v), alternatively about 0.18% - about 1.4% (w / v), alternatively about 0.19% - about 1.3% (w / v), or alternatively about 0.2% - about 1.2% (w / v), alternatively about 0.02% (w / v), alternatively about 0.025% (w / v), alternatively about 0.03% (w / v), alternatively about 0.035% (w / v), alternatively about 0.04%, 4% (w / v), alternatively about 0.045% (w / v), alternatively about 0.05% (w / v), alternatively about 0.055% (w / v), alternatively about 0.06% (w / v), alternatively about 0.065% (w / v), alternatively about 0.07% (w / v), alternatively about 0.075% (w / v), alternatively about 0.8% (w / v), alternatively about 0.085% (w / v), alternatively about 0.9% (w / v), alternatively about 0.095% (w / v), alternatively about 0.1% (w / v), alternatively about 0.2% (w / v), alternatively about 0.3% (w / v), alternatively about 0.4% (w / v), alternatively about 0.5% (w / v), alternatively about 0.6% (w / v), alternatively about 0.7% (w / v), alternatively about 0.8% (w / v), or alternatively about 0.9% (w / v).

15. The method of any one of the preceding claims, wherein the separation reagent and / or thefirst sample further comprise a labeling dye.

16. The method of claim 15, wherein the labeling dye is added to the at least one nucleic acid prior to loading the first sample.

17. The method of any one of the preceding claims, wherein the first separation further comprises rinsing the conditioned capillary with an acidic solution and / or water prior to loading the separation reagent.

18. The method of any one of the preceding claims, wherein after the first separation the method further comprises at least a second separation, wherein the second separation comprises: rinsing the conditioned capillary with water and / or an acidic solution, loading the separation reagent onto the conditioned capillary, loading a second sample comprising at least one nucleic acid onto the conditioned capillary, and applying a voltage to the capillary to effect the separation of the sample.

19. The method of any one of the preceding claims, wherein the at least one nucleic acid is selected from the group consisting of a plasmid, deoxyribonucleic acid (DNA), ribonucleic acid (RNA), single-stranded DNA (ssDNA), double-stranded DNA (dsDNA), microRNA (miRNA), messenger RNA (mRNA), DNA fragments, and RNA fragments.

20. The method of any one of the preceding claims, wherein the method further comprises detecting the at least one nucleic acid in separated sample.

21. The method of any one of the preceding claims, wherein the method further comprises detecting at least one plasmid isoform.

22. The method of claim 21, wherein the plasmid isoforms are super coiled, linear, or open circular.

23. A kit for conditioning a capillary comprising a conditioning buffer comprising a surface active polymer, a separation reagent comprising a separation polymer, and instructions for use.

24. The kit of claim 23, wherein the kit further comprises a labeling dye.

25. The kit of claim 23 or claim 24, wherein the surface active polymer and the separation polymer are different, wherein the surface active polymer is selected from the group consisting of a crosslinked polymer, a linear polymer, a slightly branched polymer, an amide, a heterocyclic polymer, polyvinylpyrrolidone, polyethylene oxide, polyethylene glycol, a substituted acrylamide, a methylated acrylamide, a polyvinylalcohol, and a dextran and the separation polymer is a cellulose derivative or a linear polyacrylamide.

Images