Originally Posted by Bassaml7
Nothing special at all, just a typical PCR.
If I add 0.5 ul to 50ul reaction volume then that means I am delivering 2.5U to the reaction volume . It's known that the optimal amount ( not concentration) ranges from 0.2 to 2 units . The number you calculated , which is 0.05 , is a concentration not an amount . And it's a rather high concentration for Taq . Too much Taq may result into smear in Gel electrophoresis due to amplification of non-specific products . 2.5 units / 50 ul may be acceptable , but , still , 0.5 ul is a small volume to deliver , especially that Taq is supplied in a solution containing 50% glycerol which means it has high viscosity which further complicates pipetting small volumes .
Thanks for this! I knew there was something I was missing when typing my response, but I couldn't remember what it was. Yeah, Taq is usually quoted in units and not concentration. My bad...
Taq is highly sensitive; the one I'm using was prescribed as 1-2.5 units. I tend to use less the lower range, which is around 1.5 units. For visualisation, I use 0.1 ul of Taq for a 15 ul reaction and have consistent results.
As for pipetting Taq, I generally put it in the Mastermix, whereby I'll pippette a larger volume. I usually leave out the dNTPs or template out of the Mastermix, which is generally in a higher amount than Taq and is easier to pipette individually. I've never had any problems with this, and I find that Taq is hardly a factor in any failed PCRs.
Erm, so basically I just think that diluting Taq is not a good idea... :P