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Old 11-17-2014, 02:14 AM   #12
nucacidhunter
Jafar Jabbari
 
Location: Melbourne

Join Date: Jan 2013
Posts: 1,238
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Hi bunce, I am fine with disagreement as far as it is evidence based and I agree with you in that cleanliness or tolerance of contamination is dependent on application and other factors. In two-step PCR usually first PCR goes for 15-20 cycles and then an aliquot is used for second PCR, say for another 10 cycles. This would generate fewer artefacts than a similar one-step PCR cycled 30 times because the amplicons would have less concentration and PCR reagents are replenished reducing the chances of incomplete extension and high concentration of amplicons two major cause of PCR artefacts.

The level of cleanliness you are hinting is more relevant to forensic and ancient DNA work which unfortunately most of the time suffers from contamination during or even before sample collection.

Out of interest is there any evidence that how many of those 10^5-10^9 aerosol copies of DNA that travel through HEPA filters and build in a lab, contaminate the work being done in that lab.
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