Contamination......what can this cause?

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micky_74
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Contamination......what can this cause?

Post by micky_74 » Wed Aug 30, 2006 9:26 am

Hello every body.
It seems that after two weeks I set up a new PCR reaction, suddenly a contamination appears. Now is the problem. I reordered other primers and I will start wrking on them, but it is possible that a contamination make your PCR not working anymore? Can you observe an optimized PCR suddenly becomes white and your white becomes dirty?
Cheers

Micky

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canalon
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Post by canalon » Wed Aug 30, 2006 2:56 pm

White is for negative control I assume or no amplification...

Many things can contaminate a PCR, and reordering a new set of primer is usually not the first thing to do. Contamination can come from any other component of the reaction, so a good start would be to change water, MgCl2 and Buffer, since they are cheap Before discarding Taq, primers and dNTP it could be interersting to test your reaction with clean reagents (borrow some from someone without problem) and try to pinpoint the contamination source, or discard if your lab can afford it.

As for your PCR not working anymore then again, plenty of things can explain that. I would check my positive control DNA for degradation, since it sems that the contamination in your negative control are pointing toward working reagents. But then it is surprising that you do not see the contamination in the positive control too. Another lead would be to have a careful look at your thermocycler. Change tube positions, the heating block maybe down...

That's all I can suggest for the moment, good luck.
Patrick

Science has proof without any certainty. Creationists have certainty without
any proof. (Ashley Montague)

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