First PCR Worked, Second PCR Did not Work

[moleculardude]

I got another good question from someone:

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. Thank you.

[eul]

Hello!

Was the weak band the one for your gene? Did you check how many bp the band was? If you are not sure check your blank sample. If there is also a band there then i suspect that you had contamination. If your blank was clear and you are not sure about the size of your band it may be unspecific product.

Did you use the same conditions in both times (Tannealing)?

If you are not sure which is the best Tannealing temperature for your primers just try a touchdown pcr and see if you get or no the same band.

Hope i helped you

[atlantide]

I would say it is "normal"... PCR is quite sensitive to your mood, the one of other people, the wind or the moon... Sometime you twice the same thing and it is not working! And it is frustrating.

I would not exclude a pipetting error, or forgetting the Taq the seconde time. Do you have a prositive control, a DNA sample which is working all the time. If it is working fine. You can be at a limite of concentration for your sample. Try a more diluted (me sometime up to 1/100 improve the PCR) and a more concentrated one.

And next time you try give a nice prayer to the PCR god, in a lab close to ours they have a specific PCR dance... I am wondering if I will try it

Caro

[danfive]

PCR god--LOL. We pray to the whole pantheon of molecular biology gods. Great to see another believer.