Posted 18 August 2004 - 07:54 PM
I was absolutely struggling for weeks with a PCR from tobacco genomic DNA.
the target fragment is 1.6kb, the primers match the target perfectly, both of them have a Tm 58. polymerase is Takara ExTaq. gDNA is well coz the positive ck always works well.
I got one "very very" weak band around 1.6kb(no other band), with some weak smear (94C, 1min; 48C, 2min; 72C, 2min; for 36 cycles.). increasing the anneal temperature(even just 50 or 49C), or decreasing anneal time(1min or 1min30s), there were no band. In lower anneal temperature, all I got was smear. The single primer PCR results showed some nonspecific bands but all of them were not around 1.6kb.
Coz the band is too weak; I try to do a 2ed PCR to enrich production, then I can digest the fragment with some RE to confirm it. I used a final 1:200 dilution of 1st PCR production as template, same primer of 1st PCR, anneal tm I tried was from 50, 52, 54...to 60C, 15 or 20 cycles. All I got was smear. What’s wrong??
I have done a ligation of the 1st PCR fragment today, if it works I can confirm it very easy. But I'm just wondering why my 2ed PCR doesn’t work????
Thanks a lot.
Posted 19 August 2004 - 01:13 AM
I have no other idea at the moment. Did you purify your DNA after the first PCR?
Did your ligation work?
Posted 26 August 2004 - 04:11 AM
You might have trouble with the thermal performance of the instrument. Lowering the denaturation temperature to 92°C might help.
Posted 26 August 2004 - 01:33 PM
maybe your template-DNA is somehow bad?
someone in our lab also had problems doing a "simple" genomic PCR (on Rice) and just got smears for all the reactions!
After extracting new DNA everything worked fine..