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Extraction of 12 bp DNA from polyacrylamide gel


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#1 gellin

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Posted 28 May 2011 - 08:25 AM

I'm attempting to extract very small (12-25 bp) DNA from a 15-20% polyacrylamide gel. Unfortunately, such high percent gels are required to achieve optimal separation of our DNA. I need to obtain at least 15 uL of fairly concentrated DNA for analysis by LC-MS. Is this an impossible project? Could I possibly see better separation with a lower % gel if I run a longer gel? Is this worth trying? Are there other variables I can change to increase the percent recovery of DNA? Also, can you suggest any particularly good extraction kits or products? Our lab has tried DNA extraction using NaOAc and subsequent EtOH precipitation with minimal success. Thanks in advance for any suggestions!

#2 perneseblue

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Posted 28 May 2011 - 04:25 PM

I don't know of any kit

The best thing I can think of is using electrolution in a dialysis tube with a 3K MWCO.

Electrolution would pull the DNA out of the acrylamide gel. The DNA will be retained in the dialysis tube . You can then move the DNA solution to a microcentrifuge tube and use a spinvac to dry the DNA sample.
May your PCR products be long, your protocols short and your boss on holiday

#3 BioMiha

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Posted 28 May 2011 - 11:57 PM

You could also try a 3-4% agarose gel to separate your DNA. It has a much lower resolution but it does its job. And isolating DNA from agarose is much simpler than polyacrylamide.




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