2 replies to this topic

[turtle]

I am genewalking using the Genomewalker kit, which works by cutting HMW DNA and ligating on an adapter, then amplifying using a gene-specific primer and an adapter-specific primer. I have noticed that I consistently get multiple bands in my PCR, and when I sequence them from the gene-specific primer, I encounter the adapter sequence at the same point in the 1200 bp band as in the 3000 bp band. Also, the sequence breaks off abruptly at this point. I'm wondering if the longer bands could be dimers formed by shorter bands annealing at the adapter. The adapter sequence is very prome to primer-dimers

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.

Thanks,
Turtle

[smesme]

Hi,

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?

regards, Søren

Søren M. Echwald, MSc., Ph.D.
-----------------------------
Exiqon A/S
Bygstubben 9
DK-2950 Vedbaek
Denmark

www.ProbeLibrary.com
One Real-time PCR kit, which covers 38.565 genes

[turtle]

Søren-
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

Turtle