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Old 11-22-2014, 12:41 AM   #17
bunce
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Location: Perth

Join Date: Sep 2012
Posts: 55
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Quote:
Originally Posted by BioKiwi View Post
If I understand you correctly, you are saying that contamination by PCR is inevitable and nothing can be done about it. I have to disagree with you because PCR has been and is and probably will be corner stone of molecular biology.

If I open my PCR tube in a PCR cabinet with outside filtered air pushing through, any aerosol will be pushed away by air flow. Any possible remaining amplicon can be degraded by UV light or cleaned chemically and cabinet will be free from any amplicon. The question is what evidence you have or you can refer to show that usual lab hygiene is not sufficient to prevent PCR contamination. An amplicon that supposedly sticks to lab wall, how likely can be airborne again and land in a PCR tube. In a very slim probability if it lands how that really is going to affect results because the target amplicons will be in billions.

I strongly, would suggest external expert review of ancient DNA set up and practices if they can not control PCR contamination.


I never said PCR contamination is inevitable - there are many systems in place both molecular - (e.g using Uracil and UNG) and physical (ART tips and airflows) that minimise its potential to contaminate.

My warning was, and still is, around amplifying amplified DNA.

Using your scenario - working inside a PCR cabinet - as an example. If you open first round PCR products (to put thin into second round PCR) inside the cabinet aerosols are still an issue within the cabinet.

As some have pointed out; contamination is dependent on copy number - but in cases where 1st round PCR's have failed to amplify then any contaminating molecules can prime the 2nd round PCR. It is a risk, and people should be aware of it.

Evidence of contamination in PCR is everywhere - have you never had a contaminated PCR? - if the answer is yes then that is evidence that "control measures" you used failed. Thanks for the offer to review our lab practices - If you want to come visit our clean lab then please do;(https://jdlc.curtin.edu.au/facilities/trace.cfm) - always looking for cleaner ways to generate data.
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