I have excised around 90 bands from two DGGE gels and put them into 2 ml tubes separately, I washed them twice with NF water and then added 50ul NF water to a level that just cover them, I put them in 4 degree overnight and then perform PCR but only the first 14 success and the others fail, I checked the DNA concentration of samples randomly and found the DNA concentration are all too low to be detected.
Could someone give me suggestions to fix the problem?
Thank you very much.
Hi - I spent a while extracting DNA from TGGE bands and struggled a bit too with some samples, there are various solutions/reasons for PCR not working. It is unclear to me how you proceeded to extract the DNA and amplify via PCR - could you perhaps clarify the procedure you used and I may be able to help?
Thank you for your help, I just let the bands stay in the NF water in 4 degree for around 12 hours and did not do anything, I asked someone and they said I may cut the band into pieces and let it stay in the water for a longer time, what other solutions you think would be useful? Thanks.
p.s. I actually cut the bands out from the gels twice and last time I used 0.2 ml tubes to hold the bands, and only PCR of the first 10 bands extraction works but not the others, so I think it is not a random error.
I think you might get a better success rate and higher DNA concentration if you physically extract the DNA from the gel band by centrifugation. I used to store the gel pieces in ~50ml elution buffer (AE buffer, Qiagen) in 2ml tubes for 12 hours (and did not perform a pre-wash). I then put the sample (gel plus buffer) into sterile spin-columns (Spin-X centrifuge tube filter; Costar) and centrifuged at 13,000 rpm for 10 minutes. I then removed the stain (see below) and used 10ul of the resulting eluate for the PCR.
If you stained your gel with e.g. Cybergold, you will need to remove the stain via ethanol precipitation prior to PCR, as the stain may inhibit your PCR success.
Hope this helps - good luck!
You have single stranded DNA (sensitivity to nucleases is high) in there - and be sure you remove (dilute) denaturation agent
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