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[Biologystudent]

It's my understanding that due to the 3'-5' exonuclease activity of the Phusion polymerase, the PCR-product will NOT have any A-overhangs. Meaning the PCR-product will have blunt ends.

So in order to follow the experiment up with TOPO-cloning. I need to create A-overhangs. Has anyone done this setup with Phusion-TOPO and can give some feedback/advices?

 

 

1) Phusion PCR (already done and stored in -20C for 4weeks)

2) Purify the PCR-product from remaining Phusion polymerase/dNTPs/primers/etc

3) Add 0.7 - 1 unit Taq polymerase (not necessary to change buffer)

4) Incubate 72C, 10min

5) Use directly in the TOPO cloning system

 

All steps performed on ice (except incubation)

Should I be concerned that the PCR-product (that is ALREADY made, but has no A-overhangs) have been in the -20C for close to 4weeks? Would that affect the results?

 

My supervisor doesn't want me to make any fresh PCR-products. Instead I use what I got. And also the simplest would be to generate a new PCR-product using the Taq-polymerase (hence no need to artificially create the overhangs alter), but that would require to buy the kit and I guess the supervisor once again wants to use what he already has in the lab.

 

Thanks!

[almost a doctor]

Your approach sounds good but is missing a couple of important things.   In order to add A-overhangs you need to have As in the mix

 

To your purified PCR you should add dATP, Taq buffer, and Taq polymerase.  I don't understand your note of "not necessary to change buffer", your PCR product will most likely be in TE or water after purification, so you need some PCR buffer back in for the Taq to work. 

 

I don't think the fact that your product has been at -20C will be a problem.  I also don't think you need to perform all steps on ice.