making hot start TAQ - (Apr/17/2007 )

Hi I have been trying to chemically modify TAQ for a few months now with no great success

I was just wondering if anybody on the forum has done it or even attempted it,
because at this stage any advise would be very helpfull to me...

thanks alot

well never tried chemically modifying Taq before,
the closest thing I have hear of is a pseudo hot start taq protocol. Basically in this protocol, one makes the PCR mix as usual but the taq is excluded. The mix in then place on the thermal cycling block, and heated to 94 Celsisus, at which time, machine is paused and the taq added. The PCR reaction is then allowed to resume.

But is hot start Taq really that much better then normal taq? Since Taq is the last component added to the mix (which is kept on ice), and the thermal cycling block ramps up to 94 Celsius rather fast, it doesn't look like Taq has much of a chance to miss prime

I agree for normal PCR reactions the advantage isnt huge...i wouldnt even use HS taq in a normal PCR!!!
in our lab we spend alot of money on TAQ gold for our multiplex reactions
so my boss asked me to make my own hot start

It seems nobody else has tried this, so let me be the 1st to say..."its difficult " and
I can see why it costs so much more than normal taq!!

Another thing I've discovered is that TAQ is not a baby...this enzyme can take quite a hammering
and still work great, if i mentioned the things ive put it through and chemicals ive added to it...you would
cringe!!!

thanks anyway