Purification of nucleic acids
a technology of nucleic acids and purification steps, applied in the direction of sugar derivatives, organic chemistry, etc., can solve the problems of human intervention, time and expense, and the centrifugation step is not automated, so as to save time, money and manual labor
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example 1
Evaluation of an 80 μL Bed Volume Pipette Tip Column Containing a Silica Resin for Purification of Plasmid from Eukaryotic Cells
[0098]In this example, the performance of 80 μL bed volume pipette tip columns is evaluated. The pipette tip column is constructed from a 500 μL pipette tip (Tecan) and is packed with a silica-based particle resin. These columns, buffer conditions and column processing procedures are tested for the recovery of DNA plasmids from complicated samples. The yield and quality are assessed by UV spectrometry and agarose gel electrophoresis.
[0099]Samples are prepared by first growing a single yeast colony in 25 mL media supplemented with the appropriate carbon source and antibiotic to propagate cells containing the DNA vector. The liquid culture is incubated at 30° C. with shaking until the media becomes turbid. The culture is divided into equal aliquots.
[0100]To purify the DNA plasmids from the lysed yeast cells, the pipette tip columns are processed by the ME sem...
example 2
Purification of Plasmid DNA from E. coli Using the PhyNexus ME or MEA
[0107]Columns and methods for purifying plasmids DNA from E. coli lysate were developed for 12 at-a-time sample format. The method was designed to operate on a PhyNexus ME and MEA Personal Purification Instruments and other robotic liquid handlers. The procedure used was as follows.[0108]1. Add 150 μL of Lysis Buffer containing RNase to 350 μL of cell culture containing plasmid. Mix using gentle pipette mixing with wide bore 1000 μL pipette tips for 30 back-and-forth cycles of 1 mL / min. On the final dispense, blowout an extra 2 μL. Wait 5 minutes.[0109]2. Add 210 μL of Precipitation Buffer to lysed cells using gentle pipette mixing with 1000 μL pipette tips for 8 back-and-forth cycles of 1.2 mL / min. On the final dispense, blow out an extra 2 μL.[0110]3. Discard pipette tips and attach plasmid DNA pipette tip columns to 12-channel head.[0111]4. Setup a 96-well microplate consisting of 1 mL, pyramid-bottom wells with...
example 3
Plasmid Purification from 150 μL E. coli Cultures
[0132]Columns and methods for purifying plasmid DNA from E. coli were developed for 96 at-a-time sample format. The method was designed to operate on a Tecan Freedom Evo® or other robotic liquid handlers. The columns used in this example were 80 μL bed columns containing silica resin and fitted with 100 μm pore size screen bottom frits. Columns were packed with or without a top screen frit of 100 μm pore size. The top frit was positioned either a fraction of a mm or several mm above the column bed. The procedure used was as follows.
Growth of E. coli Liquid Cultures
[0133]Three different growth media were evaluated. A test tube containing 5 mL Luria Broth (PML Microbiologicals, Cat. #B8474) culture, supplemented with ampicillin to a final concentration of 100 μg / mL was inoculated with a single colony of DH5α harboring a pCR2.1-TOPO cloning vector (Invitrogen, Cat. #K4560-01) grown on an LB agar Petri dish supplemented with ampicillin to...
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