PCR positive then negative result

[moleculardude]

Dear all,

I have designed a set of primer pairs for a particular gene. The first PCR result showed positive result for this gene but the band was weak. A second attempt on amplifying the gene with the same set of primer pairs did not generate any bands at all. Would like to know the reason why a positive result on the first attempt but a negative outcome after the second try of the PCR.

Confused...

thanks!

[yucel]

Hi,
Did you see the primers on gel? And maybe your primers degraded, make new primer dilution and check again.
How is your DNA or cDNA quality. It' better also check this. ıf ıt's all normal, you can try to make PCR by decrease 1-2 C annealing temperature.
good luck
yücel

[moleculardude]

Thanks for the tips,
I think I need to check my primers again.

Cheers Yucel!

[dstatay]

Hi mate¡¡

I think that your sample maybe could be degradeted. If you use this sample several times or is a old sample the DNA became to degradate to several cycles of freezing. Try to get a new sample, measure the quality and performe your PCR.
Maybe you must change your conditions; you can taste with several annealing temperature.

It´s all friend.

Good luck.


I have designed a set of primer pairs for a particular gene. The first PCR result showed positive result for this gene but the band was weak. A second attempt on amplifying the gene with the same set of primer pairs did not generate any bands at all. Would like to know the reason why a positive result on the first attempt but a negative outcome after the second try of the PCR.

Confused...

thanks!
[/quote]

[mia]

hi,
I think you may try to repeat the PCR with the same conditons as the first time (primer concentration, same sample, thermal cycler, etc). if you don't get result, try to decrease the anealing temperature 2 degrees.

good luck .

[richadeo]

I think u should check ur primers on Polyacrylamide gel using silver staining. If primers are fine then u need to alter other conditions.Try playing with annealing temperature.
Best luck.