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PCR Problem: Inconsistency with the results since one week


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#1 Sandratchats

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Posted 01 February 2011 - 07:07 PM

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

#2 ivanbio

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Posted 01 February 2011 - 07:12 PM

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.

Ivan
Carlsbad, CA




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