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PCR Problem: Inconsistency with the results since one week - Inconsistency (Feb/01/2011 )

Hello Guys, I really need your help. I am using at the moment 4 cells lines (MCF7, MDA MB231, UACC, Female DNA) as controls for my PCR. I did a PCR reaction the first time with those cell lines (Annealing temperatur: 58). I was able to see a product for all the four cell lines. I repeated the PCR with the same cell lines and same conditions, but I wasn't able anymore to see products for MCF7 and UACC like the first time. I only saw PCR dimers for the two cell lines. The third time, I got different results: I wasn't able to see any products, only primers dimers. What could be the reason? Please help me.

Thank you so much


This sounds to me like your primers are not being stored properly, or you are freezing and thawing them too often. If you are not already doing it, I would suggest aliquoting your primers and freezing the aliquots at -20oC for storage. When you need to use the primers simply thaw one aliquot instead of your whole primer stock. Also, I would recommend storing your primers at a high concentration (like 100 micromolar), and only diluting them right before using them in your PCR reaction.

Good luck.