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
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?