Site Directed Mutagenesis - Stratagene's Quickchange II XL Protocol - Oligo Issues - Melting Temperature (Nov/24/2010 )
I am using Stratagene's Quickchange II XL site-directed mutagenesis (SDM) kit to introduce a base substitution into my plasmid (15kb), however I have found some issues with the estimated Tmof the oligos.
I used the Agilent website to design some oligos and ordered them from Integrated DNA Technologies (IDT). The specification sheet that came with the delivered oligos stated that the oligo TM was 64degC. However, when I originally designed the oligos on the Agilent website, the Tm was quoted as being 78degC. The Quickchange II XL protocol states that the oligo Tm must be at least 75degC.
I have since tried to design alternative oligos, experiencing the same outcome, whereby Agilent estimate a Tm of 78degC and a Tm-calculating equation gives a Tm of 66degC.
Does anybody have any idea why this disparity exists i.e does Agilent's website take other factors into consideration, such as NaCl concentration of the final reaction mixture? Most importantly, would you advise that my SDM will work with these primers (GC = 47%, length = 34nt).
Any help would be hugely appreciated.
The calculated Tm's vary widely depending on the algorithm and the assumptions used about buffer conditions. Ignore them. You designed oligos with the program from the vendor of the kit. They know how to do this. Trust them.
Far too much attention is paid to the Tm's of oligo, and far too little to trying things and just getting on with it.