Problem with Genome Walking sequences results

[IMRPR]

Hello everyone My question is about a problem I'm having finishng the sequence of a gene. I'm using the Genome Walking Kit from Clontech for this process, the PCR products from genome walking are inserted into the vector pCRII and sequenced'm with the primers M13F and M13R from the vector. The problem is that in the results of the sequencing I can not find the known sequence of my gene segment (from where the reverse primers were designed), but I find the adapter kit GW (the forward primer is complementary to this adaptor which is ligated to digested genomic DNA before the PCR reaction). In the secuence I have both ends of the vector, which discard the possibility that the segment inserted is not completely read. If contamination would not have the adapter sequence of the kit. Anyone know anything about this? Thanks!!!

[JennYeap]

Its possibly due to non-specific binding of your reverse primer. That's why its necessary to re-PCR again with nested primer using the 1st PCR product as template. Its very crucial on primer design when using Genomewalker. You can try to blast your short primer sequence against accessible databases, to confirm the primer is specific before 'walking'. Good luck

[IMRPR]

Hi! Thanks for the advice. Actually I’m repeating the process. The product that I cloned previously was re-PCR product using nested primers :/ Any way I repeated it using fresh DNA to construct new libraries. At least the electrophoresis gel for the digestions of gDNa looked completely different as well the 1st PCR and 2nd PCR using nested primers. I’m hoping for better results this time, I’m doing the transformation today… wish me luck. Thank you again.