Primers stopped working!? - PCR Primers (Jan/11/2011 )

Hello,

I've been genotyping mice tail DNA using a set of primers. They were working fine till last month. I was trying to genotype them last week and it did not work, I changed the dNTPs, made a new dilution of primers(10um) from the stock(100um, always stored at -20C), used nuclease free water. But I do not understand whats the problem. It could possibly be the primers. I do not see any bands on the gel. The PCR clearly didn't work. I ordered a new set of primers. My question is how can primers go bad all of a sudden? They are suspended in TE. Did this happen to anyone else?

Primers typically do not go bad all of a sudden, but they do have a shelf life. How long have you had these primers? Did you use the same primers from the -20oC every time? In other words, did you freeze and thaw the same tube multiple times? Primers can withstand freezing and thawing a few times, but after 4 or 5 times the primers will indeed be completely degraded and you'll get the result you got. The best way to get around this is to make different aliquots of your primers, store them at -20oC, and only use each tube 3 or 4 times.

Of course failed PCRs could be due to a number of factors, but at least in this case it seems like using new primers should solve your problem.

Good luck!

Primers do not go bad all in one go. In fact, I have not actually seen a case where the fault is the primers.
And from what you have written, you do have stock primer solution and a diluted working primer solution.

Usually when a PCR suddenly doesn't work it is the fault of the template. Then the dNTP, then the thermocycler....

I would suggest that you run the PCR on a sample of DNA that you know will give you a signal.
I have seen peltier of a thermocycler stop working once. You might want to note where on the heating block did you place your tubes. Find out if anybody complained about PCRs not working

Use a fresh batch of mastermix. Multiple freezing and thawing of mastermix is usually the cause of no signal over freeze-thaw of primers, as long as your primer stock is of high concentration. I keep my stocks at 100uM and haven't a had problem with freeze thaw.

perneseblue on Wed Jan 12 01:38:25 2011 said:

Hi, this is probably extremely late, however I can tell you that using TE can definitely be the problem with your pcr amplification. The EDTA used for the TE is many times inhibitor of DNA polymerase enzymes. I suggest you precipate your samples and dissolve in water or just tris solution.

Good luck,
Cláudia

hvangapandu on Tue Jan 11 16:54:37 2011 said:

I'm sorry, I think this is just a myth. Do the math, instead of repeating a rumor. Your PCR reaction has a final concentration of magnesium at about 2 mM. If you have your primers at 10 uM in TE, and your final primer concentration is at 0.5 uM, then you are doing a 20:1 dilution. TE has 1 mM EDTA, so the final EDTA concentration is 0.1 mM (0.05 mM from the addition of each of two primers). So, the final magnesium concentration ends up at 1.9 mM, rather than 2 mM. This is inconsequential.