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I am running PCR reactions with different sets of degenerate primers and I want to know what should go into the master mix


My usual master mix:

  • DEPC water
  • PCR buffer (-Mg)
  • 25mM MgCl2
  • 10mM dNTPmix
  • cDNA

On ice I add my forward and reverse primers to the appropriate PCR tubes, add master mix to each PCR tube, then right before I pop them into the thermal cycler I add taq and mix.


Is this a proper way of doing it? I get product but I'm thinking maybe I can improve it by changing what I add to the master mix. For example, is it better to add taq instead of cDNA to the master mix and then add cDNA before I start the PCR? Is it okay to add both cDNA and taq to the master mix together?

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1 Answer 1

up vote 2 down vote accepted

I always add the Taq to the mastermix. First it makes the handling easier and it avoids pipetting steps which can cause contamination and can also be forgot. Then the enzyme is very stable and will even tolerate room temperature without problem. Since we are going to heat this 30-40 up to 95°C, so this is clearly not problematic. Since the reaction mix is cooled, you reaction will not start. And even if it does, there wouldn't be much amplification due to the low temperature.

I usually add everything which is the same for all reactions to the mastermix (this can include the DNA as well). If I use more than one DNA I split the mastermix in half. This way you are not going to make different pipette errors for the template, which is especially important for realtime-PCR. I usually dilute the primers slightly so I can pipett bigger volumes (simply use a part of the water used to balance the PCR reaction). You can also premix primer pairs, if you want, I have never seen problems doing this.

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