RF-Cloning.org Forum

djackson

Member

Registered: 2014-01-23

Posts: 1

Extension time prediction for 2° PCR

Hi Steve,

First of all thanks for making this online resource available for the community - I discovered RF cloning just recently, and have already made several constructs that were giving me heaps of trouble using traditional digest+ligate methods. I'm a total convert now :-)!

I have a question and a comment:

Question: Am I missing something regarding the predicted extension time for the 2° PCR? For example, I have a project where the final construct will be 9788 bp, and the predicted extension time from the RF web tool for the 2° PCR is 3:13. I have used Phusion Taq from NEB before, and have not found their claim of its speed to be that accurate. I therefore stick with the old school estimate of 1 min/kb (which works well for me). Is the RF cloning website prediction based on the claimed sped of Phusion Taq?

Comment: A couple of constructs were giving me trouble at the 2° PCR stage. After some empirical optimisation it turned out I needed to significantly reduce the amount of mega-primer in the reaction. Would it be worth suggesting in the website that users should perhaps try a range of mega-primer:template ratios in the 2° PCR? After a quick gel check one should quickly see which reaction could then be DpnI digested and used for transformation. I think that'll be my standard practice from now on!

Cheers,
Dan

Steve Bond

Administrator

Registered: 2014-01-23

Posts: 130

Re: Extension time prediction for 2° PCR

Hey Dan,
Thanks for the kind words! It's really encouraging when I hear from people that are using the site.
To your questions/comments:
Yes, the predicted time is based on Phusion/iProof (50 bases/sec). If you've been having better success with longer extensions, then of course, keep doing it! It's been a number of years since I settled on that particular value, and things just sort of worked out for me, so I don't have a nice collection of projects of varying size at varying times. I wonder if anyone else out there has played around with different extension times?
That's great info regarding the megaprimer conc. Would you be willing to share the details of one of your optimization experiments along with the check gel? I'd be more then happy to add it to the Q&A.
Take care,
-Steve