Taq / Phusion mix - (Apr/25/2014 )
I want to produce huge amounts (20 µg) of several PCR products, each about 2.5 kbp. I have Taq-polymerase and Phusion-polymerase available. Using just Taq might bring up mutations, using only Phusion is quite expensive. Has anyone tried to mix both enzymes? If yes, which buffer can I use and what cycling conditions?
Thanks for your help!
If I was in your situation I would clone each of the products and do some midi or maxi preps, then digest out the region wanted. It would save you a whole heap of time and money.
Thanks for your answer, Bob. Actually this was also my first strategy, but I lost too much of the product during gel extraction. I used the Qiaex II gel extraction kit and I ended up with about 2 µg.
Yes, gel extraction is very inefficient, but if you did 10x the number you would have your 20 ug... It should still be faster and cheaper than doing the PCR. Another option for extraction is to do an elution - basically cut a hole in your gel and run the product into it, then suck out the liquid. Requires a bit of optimization and means you can't have any buffer above your gel, but should give you higher returns.
Note also that digesting 20 ug of plasmid will give you less than 20 ug of insert in a ratio that is proportional to the length of the insert divided by the length of the empty vector.