nested PCR help needed!

[micky_74]

Hello
I´m sure here people can answer......
I´m inserting a mutation in to cells (4 consecutives nucleotides in the genomic DNA). Now the frequency of cells affected is extremely low (1 every 5000, maybe lower.) Screening method: nested PCR. First I made a PCR for 30 cycles giving me an external product of 350 bp. Then I use a specific primer and a forward internal giving me a product of 123 bp. Both PCR last 30 cycles. I got some positives results (all the semples showed 3 higher bands, but only few of them a 4th band at the expected size). Where this method could be wrong? What can I do to improve it?
I went down (with serial diluition until 1 on 250 cells), the positive band is more and more bright on a gel, whils the other bands are disappearing (in the positive wells). Why can I switch to a single step PCR? And if the single step PCR is negative, means I´m completely wrong? I´m using a plasmid as positive control, but maybe the genomic region is much more complicated and I suspect that the gene I´m working with is a multicopy gene or several related sequences are presnt in the genome. Any idea on how to deal with?

Thanks!!

[luna-celeste]

Hello,

How did you nest your PCR, could you add a figure with the position of the region of interest?