Thanks to everyone for the responses. That Qiagen article was surprisingly helpful, though it would be nice for them to share the constitution of their "Q Solution".
In particular I was interested to read about the ammonium ion effects and the effects of high temperatures; the high-temperature depurination issue may have been behind some past PCR failures of mine. Good to know! Thanks again, Cathal On 10 July 2011 15:28, chovek69 <[email protected]> wrote: > 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 > -- letters.cunningprojects.com twitter.com/onetruecathal http://www.indiebiotech.com _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
