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researcher40 02-17-2011 02:38 PM

PCR contamination
How is it possible to contaminate the taq polimerase in a PCR reaction, if the taq is the last component that should be added in a PCR reaction?

luisillo 02-17-2011 05:56 PM

Re: PCR contamination
Handle issues. I'll give you the ones that, to my point of view and in my experience, are more likely to contaminate your Taq pol:

- Small drops of the enzyme buffer (in which the enzyme is diluted) can remain in the cap of Taq pol tube. There a chance of coming in contact with it, increasing the risk of contamination, if you do not spin (short centrifugation) the tube before opening it.

- Your tip may be sterile, but how about your pipette? Taq pol is usually required in small volumes, so you ought to use a small pipette (i.e. 0.5-10 ul) which use the small tips that are short in lenght. This issue makes you insert the pipette further in the Taq tube and if you're not careful enough and the surface of your pipette comes in contact with the surface of the tube, you could be contaminating the enzyme.

- Other minor mistakes like reusing a pipette tip, touching strange surfaces with the tip, touching the inner surfaces of the Taq tube or its cap or even leaving the tube's cap near contamination sources.

Something recommended to avoid reagents contamination is doing the master mix before handling the samples.

Hope this helps you

Sharda Bhagchandani 03-27-2011 08:01 AM

Re: PCR contamination
If talking about PCR contaminations, a contamination can be intrinsic or extrinsic. It can come from any source, however careful handling avoid extrinsic contamination but intrinsic can be taken seriously specially in case of diagnostic tests. Better is to use PCR reagents in aliquots.

Nickdn 04-21-2011 02:29 PM

Re: PCR contamination
I have the same problem right now. Disposed my master mix en started all over, luckely got rid of my contamination

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