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RT PCR contamination.

Discussion of all aspects of biological molecules, biochemical processes and laboratory procedures in the field.

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RT PCR contamination.

Postby georgewiggins » Thu Sep 06, 2012 9:49 pm

I am running a reverse transcription PCR (cDNA synthesis) and am getting contamination.
Currently I run two negative controls, a -RT and a -template control, both of which are firing.
I have changed everything (New; DTT, dNTPs, Primers, water{Sterile MQ}, RNase out, buffer) and have not found the contamination, I have confirm the negatives are firing by endpoint PCR with primers designed to span an intron (therefore I no there is cDNA/RNA in my negatives). I have subsequently run a negative with -template and -RT and that still fired. Thus leading me to believe there must be contamination of a previous sample (or something along the similar lines).
There have been a few other we experiments that I have tried that have improved this situation. My supervisor suggest maybe there is contamination of the amplicon I am testing. We use b-actin as a control for a lot of experiments so it is plausible but I find it difficult to believe when in my endpoint PCR I got the negative for that experiment to be negative, I figure if the amplicon can contaminate my cDNA synthesis then it has the same potential to contaminate my PCR.

Any suggestions on what I may be doing wrong? Could there something wrong with my technique?
I am currently clean very vigirously (bleach, H2O2, UV and ethanol), and will run another PCR with a different primer set to test the theory of my supervisor.
georgewiggins
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Postby Cat » Sun Sep 09, 2012 6:08 pm

Investigate tubes, tips, pipets, and such for contamination. Obtain (make/borrow/buy) new controls and run them along with the old ones. Use all new reagents in case it was pipet contamination and your “new” reagents are already contaminated…
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Postby triton » Thu Sep 13, 2012 11:05 am

what is an organism you are working with? is it present everywhere, like bacteria, or do you work with something rare like endemic mice of Madagascar? Did you consider contamination of your pipets? Do you use tips with filters?
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