Hi, currently we are having problem in recovering the DNA sequence from DGGE gel. The sliced dgge gel (DNA bands) was smashed and boiled at 55°C for 45 minutes and followed by elution with 50uL TE buffer. But, no amplification can be detected in agarose gel when the PCR was performed. Is there any other effective techniques can be utilized rather than using smashing and boiling method?
Well we have not used DGGE gel, but have performed RFLP gels (polyacrylamide gels) and CSGE gels. We have successfully amplified the band of interest from these gels. What we basically do is sliced the band in the gel and suspend the gel piece containg the band in TE (50microL) and allow it to sit there for over night at 4degC. The DNA bands does come out in TE. Next day one can perform PCR.
Usually we perform perform in 3 sets, 2microL, 5microL, and 10microL of the eluted band in TE as the input DNA. This method has worked for us, it might work for you as well.
Best of luck.
Is it possible that you elute not only DNA but also PCR inhibitors from the DGGE gels?
What do you use for denaturing in the polyacrylamid?
I agree with the previous colleagues that the best way to do it is to place the cut piece of gel in water or TE and let to diffuse it at 4°C.
The problem of the DNA degradation is relevant and for this reason sometimes the transilluminators do have two intensities to use. When you cut the bands the lowest intensity should be used.
PCR reactions using 1 ul of eluted DNA should be successful.
Instead of boiling the gel ,I did frozen-thaw 3 times in TE buffer. And used 1ul of supernatant for PCR. It worked better.
I agree that the most successful method is to carefully cut out the target DGGE band, with minimal UV exposure and resuspend in TE buffer and refridgerate overnight, using the supernatant as DNA template in the subsequent PCR. I agree with David Walker that using primers for the Post uct-out PCR that do not contain the GC clamp could be useful as I have found optimising PCRs with GC clamps attached to primers more troublesome in the past.
Make sure you are cutting out enough of your target band and maybe try a 1:10 dilution of your supernatant if the recommended method does not work incase inhibitors from the DGGE gel are causing problems with your PCR.
It may also be that some of the bands you are targeting are not real bands, but artefacts that might be appearing alongside another real band. Try cutting the same band out from different samples to see if it is something real that you can amplify from.
Hi all, some of the samples can be amplified by using smashed and boiled (after sliced and suspend band of interest in TE buffer at 4 degC over night) method. however, other sets of samples can not be amplified by using the same method. i also carefully sliced the gels under minimal UV exposure. Based on the trouble shooting mentioned above, i am afraid the problems is caused by the inhibitors. i will try to use the recommended methods. hopes it will be a success, thank you.
Hi all, Thanks for your valuable suggestion. I have cut the DNA and eluted in PCR grade water. Is that ok ? Also i need to do PCR for the cut -DNA bands. Should i use the same primers which i used for the DGGE PCR but without GC-clamp ?
Thanks a lot in advance!!!!