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Some problems of DNA recovery from native PAGE gelModerator: BioTeam
2 posts • Page 1 of 1
Some problems of DNA recovery from native PAGE gelMy 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.
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?
2 posts • Page 1 of 1
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