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add template first or Syber mix first to reduce contamination in qPCR? - (Jan/27/2013 )

Hi, I have long been confused with contamination problem during PCR.

Now I plan to do qPCR. I want to ask for the 96-well plates, should I add templates in each well, and then add syber mix? Or if I should add syber mix first, and then add template in each well?

Thanks for your suggestions!


In my view, you can add anything first, but you should be careful and not mix the tips. Just change the tip after every addition. The only thing is that we add enzyme in last.


I would definitely make a master mix (including the enzyme) and aliquot it into all 96 wells with the same tip(s). I would then add the template individually to each well with a different tip each time.


I add template first to side of each well (new tip each time), then Sybr green master mix to each well (same tip, shoot solution into well - not as bad as it sounds). Then I spin down the mixture (3,000xg for 3min at 4C) to remove any bubbles. I add the enzyme last because I have noticed a difference in my amplification plots if I add master mix and allow it to sit a RT while I am adding my template cDNA. I may be paranoid, but it works for me.