Finding PCR Limiting Reagent - (Nov/09/2005 )
I am trying to figure out how to calculate which "ingredient" will run out first during my pcr. This is based on a 3500bp pcr w/ GC of 57%. Standard, 50X dNTP used, etc. I was having some errors with this amplification and I wanted to see if perhaps some of my dNTP's were being completely used up, or it was the taq, etc. I have searched for calculations and I know I am just missing something simple, but I would like to know if anyone has a quick answer as to how to figure out this equation/problem. Thanks for any help.
No answers? 
If you need more information just let me know.
The only component that you will "run out of" is the Taq. When the number of molecules of template exceed the number of molecules of Taq, the reaction goes into plateau phase. Doubling the amount of Taq will only add one additional cycle before plateau.
25 cycles of PCR to amplify a 500 bp target will only consume about 4% of the dNTPs according to ABI.
25 cycles of PCR to amplify a 500 bp target will only consume about 4% of the dNTPs according to ABI.
Thanks for the information. Is there a document where ABI says this? So doing 35 cycles of a 3800bp would still come no where close to running out of dNTP's. The taq I was using had proofreading functions. Can that kind of taq become sloppy after that many rounds? I was having purine transitions, which is why I thought I might be running out of dNTP's, but the taq might not accurate enough after that many rounds too.
The purine transition has nothing to do with running out of reagents, but rather with the fact that there is a deamination of your dCTP's to dUTP after extended time @ high temperature. Pure proofreading enzymes will incorporate dUTP and no longer exhibit polymerase activity, but taq itself just keeps going.
More info: Hogrefe, H., et al. ( 2002), Proc Natl. Acad. Sci. USA 99: 596-601
or even better: Available for free
More info: Hogrefe, H., et al. ( 2002), Proc Natl. Acad. Sci. USA 99: 596-601
or even better: Available for free
Vairus,
Thanks for the paper, most of my errors were the G to A, which would be explained by the dCTP deamination, although the other half of the errors are A to G, which should not be attributed to a deamination. However, this gives me info I need to try and fix my problem and get these covering plasmids amplified correctly.