product dimers with GenomeWalker kit
Posted 22 September 2004 - 11:21 AM
Has anyone else encountered this with this kind of gene walking? Is there a solution?
I have one fragment which appears to contain an extensive inverted repeat. How do I tell if this isw for real, or a result of a dimer? I can't find the adapter sequence, but there is a lot of slippage, and long runs of single nucleotide repeats.
Posted 23 September 2004 - 12:36 AM
it sounds tricky.
If the sequence breaks off, but the fragment is longer, it is usually because there suddenly occur many different sequences in the same band, hence the signal is blurred. this could well indicate that the adapter sequence is fishing out other sequences to add on the end.
I once tried the old fashion inverse PCR, which is similar to what you do, just without the adapter. Using such a parallel method might answer your question.
I guess an old fashion Southern blot might tell you if the sequence is real?
Søren M. Echwald, MSc., Ph.D.
One Real-time PCR kit, which covers 38.565 genes
Posted 23 September 2004 - 08:07 AM
You mention inverse PCR. What do you use as the second primer? I am familiar with reverse transcription (mRNA to cDNA) where one uses a poly-T oligo. Is this what you are referring to? I'm working with genomic DNA, so it isn't an option here