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How to Use Violet GlasPac-Filled MicroColums™ for High Throughput Purifying of DNA from Alkaline Lysis, Tris Buffer Solutions, Agarose Gel Slices, or Other Sources
1. Bind DNA
a) If sample volume is 50ul or less, add 150ul of #4 Salt concentrate to sample. Then, bring volume of the sample solution to 200ul with ddH20. Or, if sample volume is greater than 50ul, add #4 Salt concentrate in a 3:1 volume ratio to sample.
b) Pipette 200ul of sample solution into a BioPak MicroColumn. Pipette up and down at least 3x. If sample solution is greater than 200ul, pipette up and down in a seperate tube or well.
c) Dispense and save solution from step 1(b) in a clean tube or well.
- To maintain hydration of resin, never completely dispense buffers from the MicroColumn
until the final wash step 2(c), just before the elution step 3(a) below.
- Ideal volume of sample solution is 200ul at a 3:1 ratio of #4 Salt concentrate to DNA sample.
- If volume of sample solution is greater than 200ul, and binding capacity of the MicroColumn is not exceeded, repeat steps 1(b) and 1(c) with any remaining sample solution.
- If quantity of DNA in the sample solution exceeds binding capacity of the MicroColumn, Wash and Elute as shown below. Then, repeat steps 1 through 3 using the same MicroColumn.
2. Wash DNA
a) Prepare a Final Wash solution by adding 102ml of ethanol, and 68ml of ddH20, to the total 30ml of #5 Wash concentrate. Pipette 200ul of this solution into the BioPak MicroColumn. Dispense and discard. Repeat the process 1x.
b) To remove any residual Final Wash solution, pipette 200ul of 75% ethanol:ddH20 solution into the BioPak MicroColumn. Dispense and discard.
c) Aspirate any remaining wash solution from the tip. This can be done directly to the BioPak MicroColumn with an aspirator, or you can eject the tip from your pipettor into a BioPak VacRack with the bottom removed, thereby allowing access to all the tips with an aspirator.
d) Dispense and save sample solution from step 2(c) in a clean tube or well.
3. Elute DNA
a) Pipette 60ul of ddH20 into the BioPak MicroColumn.
b) Pipette eluent up and down several times into an empty tube or well.
c) Dispense purified target molecule into your collecting tube or well.
- Heating ddH20 to 50C, before pipetting it into the MicroColumn, moderately improves elution.
- Elution efficiency is further improved by allowing the resin to soak approximately 3 minutes after pipetting ddH20 into the MicroColumn.
- Additional DNA may be recovered with a second elution process.
- If desired elution is not accomplished, it is possible that too much DNA is being washed out of the MicroColumn. To prevent this, a more diluted wash solution should be prepared by adding 2 volumes of ethanol to 3 volumes of previously prepared Final Wash solution from step 2(a). Then, repeat steps 1 through 3 using the same MicroColumn.
Protocol adapted from Molecular Cloning, A Laboratory Manual by Maniatis, Sambrook & Frish.
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