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Preparing PCR reactions from a master mix


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#1 Julio-Claudian

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Posted 20 June 2009 - 04:00 AM

Dear all,

Been a whole week before I got my PCR optimized. Looking back, I did a lot of tweaks: MgCl2, annealing temp, denaturation temp (GC rich template), reducing primer conc (due to primer dimer). All these are explained in detail over the years.

But then I realize something, I prepared those tubes from a master mix and though it may be simple, I can't think of a reason why anyone would add the template in the individual tubes instead of the master mix. Understandable to add polymerase last but template too??

Now, I was thinking that it wouldn't be a problem to add the template into the mix since if the primer were to anneal, won't the initial denaturation separate them? Or it it because the vortexing will shear the template?

Note: Template here means the genomic DNA.

Thanks people! Have a great day!

#2 HomeBrew

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Posted 20 June 2009 - 04:56 AM

If your intention is to aliquot the mix to several individual PCR tubes containing identical PCR reactions (same primers and template), there's no reason not to do that.

If, for example, I want to do four identical 50 ul reactions to get a ton of product for gel extraction, I make a 200 ul master mix containing all the components -- including primers and template -- mix well, and aliquot 50 ul to each of four PCR tubes. It works fine, and avoids having to take very small volumes with a pipettor.

#3 jiajia1987

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Posted 21 June 2009 - 05:46 PM

To me, the only problem of adding template into the master mix is that you can get variable amount of starting template for each PCR reaction, unless you really mix them up properly and aliquot well.

#4 Julio-Claudian

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Posted 24 June 2009 - 03:12 AM

Thanks HomeBrew and jiajia1987, I'm sure we've heard many "tips and tricks" but some are really undocumented. But then again, it doesn't hurt to listen to some with the experience.




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