Problems sequencing DGGE products - (May/31/2007 )
I'm having some problems sequencing excised DGGE band, and I'm just wondering if anybody else had this problem.
I'm working with deep sea water looking at bacterial communities. I have carried out PCR (using GC clamped primers) on the extracted DNA and run products on DGGE gels. I have excised bands and cleaned them up as best as possible by running them on subsequent DGGE gels, cutting the band out again and repeating a few times. Then I carry out a PCR (using non GC clamped primers) ethanol precipated PCR products and sent samples for sequencing. But they keep failing the sequencing procedure, and I've no idea why??????
Any info would be appreciated.
We excise the bands from a gel and leave them in water overnight. The next day we mash the band in the water to release the DNA. We carry out PCR and then clean the PCR product up, this cleaned up PCR product can then be used for template for sequencing. Alternatively you could clone the PCR product and seuence from this.