New England BioLab Taq Pol - (Jul/29/2005 )
I've been doing some PCR on mtDNA of Pythium spp. When I use the Promega Taq Pol it works fine, however, using the SAME samples with the New england BioLab Taq pol the amplification does not work.
Does anybody use this enzyme before and have recomendations about the conditions to get good results?
Does anybody use this enzyme before and have recomendations about the conditions to get good results?
NEB often use their own special unit definitions - have you checked thet you are using a proper amount of enzyme?
Hi,
I'm using NEB Taq Pol, works good for me (the whole lab). I use 0,2 µl of Pol in 25 µl PCR mixture. Earlier I used 0,5 µl, but works really good with less. But you have to use their Pol buffer also (2,5 µl in 25 µl).
Hi there,
Keep in mind that one Taq isn't the same as another. You can assume that both taqs differ slightly from each other. Each company has its own version so to say. If you have optimized you protocol with the Promega taq there is a very good chance that if you only switch taqs you will get another result, sometimes better, mostly worse. To be sure about the performance about the NEB Taq you'll need to do an optimization experiment. You can try altering Mg2+ concentration, # taq units,... You'll probably have perfect results again in no time but with a slightly altered reaction composition. You might also want to look into witch product exactly you were using from Promega. I know for instance that NEB just gives you Taq with no Mg2+ already in there, you'll have to add it yourself.
Is MgSO4 the same as MgCl in terms of activity in PCR? ie: are they interchangable?
I'm not absolutely sure, but I think it's specific for the manufacturer. It's the Mg2+ ions that are active, but I'm guessing that the Cl vs SO4 would have some differences on the activity of the Taq, even just by changing the buffer chemistry. Some Taq's use Mn2+, so I'm guessing that they're not interchangeable at all with Mg2+. (I could be wrong tho... anyone?)
As said above, each manufacturer has thier own Taq, and their buffer is specific for their enzyme, I wouldn't go swapping it willy-nilly.
Nicole
Silly question,
Is it possible to manual hotstart with any taq pol? Or only specific types? ie: with NEB thermopol + DNA taq pol?
I beleive that you can manually hotstart with any enzyme. Just hold everything on ice, start your PCR-program and put in your tubes when temperature of the machine is high enough.
As for MgCl2 of MgSO4. I once tried to do a PCR with an enzyme that normally has the SO4 and changed it for Cl2, and I had a drastically reduced yield, specificity and all that were okay. So depending on your needs (that is if yield is important or not) you can interchange them I think.