PCR to get 10kbp product - (Jun/17/2013 )
I have to get 10kbp PCR product.
I have 2 primers, which have Tm of 58oC (F) and 60oC (R). I intend to use Pfu Taq pol to get the high accuracy.
What should I do to get success?
Thank you so much for your suggestion!
I have used Pfu to get a 7.5Kb product, no problem. I have included a link to the Pfu polymerase PCR reaction setup. With longer PCR products you need to be aware of the GC content of your gene of interest. For example, when I was cloning my gene, my product would not amplify without the presence of GC enhancer (my gene had high GC content ~60%, with alot of GC repeats). I would follow the included protocol and add the highest amount of dNTP's (1uM). If you have a gradient PCR machine, I would recommend trying a couple of different reaction conditions. Maybe one with higher dNTPs incase they are exhausted in your reaction, one with GC enhancer, possibly a couple of different annealing temps. You shouldn't have any problems. Good luck.
As you mentioned, the Elongation step you used a period of 15minutes, is it?
And the GC enhancer you mentioned is located in the target gene or somewhere else???
I would recommend Phusion or Q5 polymerase, and an extension time of only 3 minutes. Make sure you follow their cycling temperature recommendations.
Thank you very much for your suggestion. It really makes sense to me.
However, have you ever tried with Q5 pol for amplifying large fragment, say > 10kbp?
Thank you again