Taq polymerase dilution? - (Sep/21/2005 )
hi again. I just want to ask if the Taq polymerase which is normally supplied as 5,000 U per ml should be aliquoted and diluted in water before adding to pcr mix?
How can i compute it if i need .5U of Taq in a 20 ul reaction with 5,000 U per ml of Taq.
5000 U/ml means you got 5U/µl.
So, if you just need to do 1 reaction, you would need 1/10 of a µl, which is nearly impossible to take accurately. I would pre-dissolve in the same buffer your Taq itself is in. You should find the recipe on the information sheet from your supplier. Otherwise, if you're doing multiple reactions at a time, just make one master mix with the correct amount of taq. I succeed in doing PCR in a 15 µl volume for multiple reactions I need about 0,6 µl taq which I can take accurate enough (it seems by the succesful result of my reaction).
Year, measuring 0.1ul is almost impossible. Eppendprf, for example, provides several tips that may measre small amounts. They are better than ordinary yellow tips.
To be accurate, you may want to dilute Taq first, but may be not necessary unless the concentration greatly affects the reaction.
You should never be doing just one PCR (what about your controls ), however, unless you are totally poverty stricken just make a separate 1:10 Taq dilution using the smallest amount you can pipette accurately (0.5 µl for me) and throw the rest away.
Reduce DNA sequencing errors