Why is it necessary to add Taq DNA polymerase last during PCR? - (Mar/30/2009 )

Hi all,

my query as above.

Thanks

It's not necessary. Think about all of the master mixes widely used, that have Taq already present. It is a good idea to wait until buffers are present before adding enzymes, although I'm sure Taq can deal with pure water. There can be issues with premature activation of the enzymes, but keeping things on ice will solve this.

phage434 on Mar 31 2009, 10:18 AM said:

Hello,

I agress with phage434, It doesn't really make a difference when you add it, specially if the reactions are on ice. I think the only reason why people add it in the end is that the volume of the enzyme is the lowest, so you can visually put the enzyme directly into the solution and mix it nicely (it won't stick anywhere toi the tube and thus u won't loose out the enzyme).

Atleast that is the reason I add it in the end (lowest volume goes in teh end).

Best,
TC

Nowadays the TAQ doesn’t need to be added last.
A large part of them have an antibody attached to them that prohibits the TAQ from binding to the DNA (hot start). The 95C at the starting denaturants the AB and the TAQ can start working.

To the answers from Yahoo,
"If you add your DNA polymerase first, there is a chance that it will begin to work before you add your reaction to the thermocycler. The DNA will not have been denatured yet, nor will your primers have annealed. It is possible that this could result in non-specific synthesis, and you'd end up with lots of random fragments being amplified when you do put the reaction into the thermocycler"

This is partly true.
First, you can get non-specific synthesis but not in a large quantity because the TAQ needs a template to work on and if the DNA hasn't been denatured yet there isn't much for it to work on. Second, your primers won't amplify random fragments.

The main problem is the possible formation of primer dimmers. The primers lineup and act both as primer and as template while depleting both primers and dNTPs.

I always read the instructions, then think about what is really happening. If you are having problems, I'd recommend following instructions. If you need to get things done and are willing to think for yourself, then you can sometimes do things better and more efficiently. If this were really a problem, none of the PCR master mixes on the market would work, since they all contain enzyme out of the box.

As molgen mentioned, many enzymes are now hot-start, but I've never had trouble with PCR that was otherwise working due to order of addition of components. Redesign your primers -- that is almost always the problem.

phage434 on Mar 31 2009, 08:00 PM said:

Enzyme used to be some sensitive compound. It used to require some optimum conditions like optimum temperature, ph, sometimes pressure and even halo or non halo environment.

According to my observation, Taq Polymerase is added at the end because it used to be in small amount as mentioned earlier and it used to be sensitive to pH. So to give it optimum environment to preserve it for longer time in the solution....

i agree to all who have stated that it is not necesarry and all the reasons are also what i too think while doing my experiments. LAtely the inactivated plymerases have surfaced as olgen mentioned so this need not be an issue at all!!!

and Chaste, primers have to be added by us!!! (imagine universal primers for all the pcr setups ). The suppliers just provide everything else other than the primers so it is an easy pipeting event (A master mix). In fact the Mg concentration can still be altered so they do provide a stock of MgCl2 too!!