no template but still got exact size bands???!!! - PCR genotyping problem (Sep/30/2004 )
i tried to do genotyping (fly transgene in mice)
with primer sets designed from transgene.
expected product size is 200-400 bps
I use Taq
my PCR cycle is 2 min 95 degree initiation
30 sec 95
40 cycle of 30 sec 58
1 min 72 elongation total
10 min 72 final elongation
i tried 20s sample , they all showed positive results (exact size bands)
so i tried to run pcr with plasmid ,
with wildtype (wth the wildtype showed bands too! exact size !)
at final I run the PCR without template (heck , bands come as well )
I didn't do hotstart
should I go for hotstart?
or my reagent got contaminated? (so frustrated)
thanks for help
Contamination should be ruled out first. I think 40 cycles are too many.