RF-Cloning.org Forum

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