On Jul 8, 3:16 pm, [email protected] (DK) wrote: > In article > <740568f1-764d-4381-aa90-15b203e79...@x41g2000yqd.googlegroups.com>, chovek69 > <[email protected]> wrote: > > >I regularly amplify >3kb with regular Taq with (NH4)2SO4 buffer (see > >link below) + 1M Betaine and 5min extension step at 65C. > > >http://www.whitelabs.org/Lab%20Protocols/PCR%20Protocols/kyle.htm > > Just curious: why 65C? 5 min total extension for 3 kbp or per every kbp? > > Thanks, > > DK
It is 65C for 5min total ext time and betaine is critical. There are some reports stating that decreasing extension and denaturation temp could improve long PCR. See for example this http://www.qiagen.com/literature/qiagennews/0398/983pcro.pdf although there many others. I think it is because of the damage to the template during the longer cycles plus betaine could decrease the Tm a degree or two. Of course the the amplicon %GC is most important for the optimal ext temp so it is best to run a gradient optimization. _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
