How To: PCR Master Mixes

[@aleksandrkovalenko1743]

I was kinda confused why you mix primers with the polymerase in the master mix. In my experience it is rather dangerous because it can lead to amplification of primer dimers, trimers etc apart from the desired DNA template. (because we keep the mix at RT for a while but some undesired chemical processes still can occur) We are usually mixing everything but polymerase which is added at the last moment.

[@drrenard1277]

Keep temperatures low to keep from annealing. Should be safe if done within the hour or two from mixing

[@drrenard1277]

Good ole PCR. Pipette, cry, repeat

[@byankezia6798]

Where do we mixing master mix? Laminary flow?

[@indujav6897]

How is primer removed in PCR..!?

[@kalumthomson5638]

During PCR the primer anneals to the DNA strand which provides a free 3'end for DNA polymerase to begin DNA synthesis. At the end of the PCR reaction you need to then clean it up to get rid of excess primer, polymerase dNTPs etc. Various columns exist for this purpose.

[@ilyaveil3980]

I've always been taught to never vortex anything with enzymes in it, including Taq...?

[@drrenard1277]

Taq is more durable for sure but I've herd it be fine and also told to avoid. Myself, I prefer to have my auto pipette do a few in and out cycles to be safer on mix so that it's habit regardless of the enzyme