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PCR to get 10kbp product - (Jun/17/2013 )

Hi all,
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!

-DrLeo-

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.


http://www.thermoscientificbio.com/uploadedFiles/Resources/high-fidelity-pcr-reaction-set-up.pdf

-jerryshelly1-

Thanks Jerryshelly,
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???
Thanks again

-DrLeo-

I would recommend Phusion or Q5 polymerase, and an extension time of only 3 minutes. Make sure you follow their cycling temperature recommendations.

-phage434-

Dear Phage434,
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

-DrLeo-