Some problems of DNA recovery from native PAGE gel

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Some problems of DNA recovery from native PAGE gel

Post by wusilinqi » Thu May 14, 2009 5:20 am

My protocol of the PAGE gel running and DNA recovery is as follows,
1. 20% native PAGE gel (The DNA i want to purify is around 20bp dsDNA + 10 bp ssDNA )
2. running at 250 v for 30mins,
3. stain and cut the desired band;
4. cursh the gel by yellow tips into small gains;
5. add 2volume TE buffer (100ug gel ~ 200ul TE buffer)., rotating the mixture for overnight at room temperature (~24C);
6. centrifuge the mixture at 12000g for 15 min;
7. take out the supernatant, and the DNA should be in it.

The problem is that when I use nanodrop to measure the concentraion and check the purification, i found that only 10% DNA is recovered, and the product is not pure.
Please help me to find the mistakes. Thanks.

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Location: UK

Post by domwood » Wed May 20, 2009 10:25 am

Well I'm no expert on this, but I would have thought the two key steps are the physical ones, the gel and the centrifugation. Have you tried running the gel at a lower voltage over a longer period, and also centrifuging for longer.

Also what is the source of the DNA, can you be sure of the extraction method's efficiency?

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