PCR reamplifying problem - (Jun/08/2005 )
Hi, we eluted DNA (PCR product) from a gel and confirmed the results of elution through another gel run. However, since the band was weak, we decided to PCR again under the same conditions. But after PCRing a second time, we could not observe the DNA results in a new gel run. Furthermore, not even the primers show up on the new gel run. The procedure was repeated from the second PCR step and the same results were observed. What could be the problem and is there anyway to solve it?
Dear friend,
Since you could get you band from first PCR, you shouldn't not getting anything from you second PCR.
When you say "could not observe the DNA result in a new gel run" does it means totally blank/ no specific band / smearing.......?
Did you quantitate your first PCR product and second PCR producr?
Best regards
Hadrian.
hi
the mopre strange thing is that you don't observe any DNA, even the primers. That's sounds reared.
Re-amplification of a pcr product can be a tricky procedure. My advice is to do a 1:10000 dilution of your pcr product and then do re-amplification. If you have too much starting material in your reaction it will inhibit amplification. It can also lead to non specific amplication that will show up as a smear on your gel. Are you showing slight smearing? It might be even barely noticeable.
Other steps to consider while doing a dilution is:
-decreasing 2nd pcr by 10 cycles
-up your annealing temperature to two degrees
-do a step down pcr to decrease non-specific binding
Mr. Dbbiewalton is correct, is you have too much starting material, it can inhibit your PCR reaction. However, if this happen you should see smear instate of nothing.
Regards
Hadrian
Could it be DNase contamination?