PCR Reaction - (Feb/14/2012 )
Is this your first amplification or a "recipe" passed down to you?
Reason I asked is that the final concentration of Mg2+ (3mM) is quite high for me. For Taq, I usually add it into the individual tubes before starting the cycles. I add the primers into the master mix though.
I have no complains about the cycles and temperature but depending on the outcome, the annealing time
Unless this is a repeat of what was done before, you might need some tweaking after this.
Well, it is a secret recipie that is passed down to me. I'm new in technically but not in theory... So, what is the possible amount of Mg2+? 4 microlit? I'll used the master mix for 4 pcr tube... And thanks for reply.
It's good that you're familiar with the theory behind the workings of each components.
Since it's a passed down secret recipe, I'm sure it's been optimised (?). So I think there must be a reason for such values. In any case, perform the amplification, see what you get, and work from there.
I remember starting my Mg2+ at 1.75 mM and