Taq polymerase that can run 100 cycles? - (Sep/28/2005 )
I want to use a commercial taq for single primer extension. Does somebody know which taq is the best for doing PCR with lots of cycles, for example, 100cycles. I mean which company provide the most thermostable enzyme. I know it's hard to answer this question but I really need the answer. Thanks a lot.
Why do you need to run up to 100 cycles in the first place??
Look for Roche and Finzyme product.
Thanks, Hadrian. I just use one primer to extend the template. The more cycles, the more product I'll get. That's the reason.
You never need 100 cycles. The amount of product is limited by the primer concentration or dNTP concentration, not the number of cycles. In exponential amplification, the product nearly doubles each cycle. 100 cycles of doubling would amplify a single molecule to a megamole of product. You wouldn't want to be around.
yes I think phage is right. I was over look of this aspect.
During the cycling process, your product is increase, however your dNTP is depleted and also inhibitors is increases.
So the reaction might stop half way through even you programed it to be 100 cycles.
The only limiting factor in exponential amplification is the amount of enzyme. The other components are not used up. Plateau phase is defined as the point at which the number of molecules of enzyme is equal to the number of molecules of template/product. Doubling the amount of enzyme only gains you a single additional cycle and is usually not worth the cost of extra enzyme.
In linear amplification with a single primer, 100 cycles will result in approximately 100 copies of each molecule of template you start with. The limit here is the stability of the enzyme at the denaturation temperature. Taq's half-life is approximately 40 minutes at 95°C and 10 minutes at 97.5°C.
You can always add additional amount of taq during PCR cycling if needed. That is what I did when I wanted to run 45 cycles. I added new taq at 40 cycles.
100 cycles!? Yes, you can add more enzyme every now and then, but the real limiting factor here (and in any long PCR) is the kinetics of primer-template annealing, not the reagents. At high product concentration, your primers won´t anneal with the template fast enough in comparison with template-template annealing.
Thanks all kind reply. I agree tfitzwater that Taq stabilities are very important for the method. So I asked which commercial Taq product is more suitable. I've tried to add more master mixture(taq;dNTP;buffer;MgCl2;primer) after 90cycles. hope it does work.
Thanks a lot.
Your bet is adding Taq after 40 cycles.