On Jul 6, 3:26 pm, Cathal Garvey <[email protected]> wrote: > (Crossposting from Google+) > I have a plasmid that's, say, 3.7kb long, and I need to amplify it with taq > polymerase (because that's what I have, and I'm too poor to afford better > right now). It's the length that's the main problem, although challenges > relating to the specifics of how to amplify it are also troubling me. > > Specifically, I need to amplify a ~3.6kb plasmid *from another cloning > vector* as a linear molecule such that either amplicon end is complementary* > to the other, because this will enhance B.subtilis' ability to take up the > DNA and recombine it into a ring-shaped plasmid once more..or at least > enhance the plasmid's likelihood of copying itself successfully into a > ring-shaped plasmid by rolling-circle-replication before it is lost. > > Taq seldom amplifies longer than 2kb, but there are voodoo ways to make it > do so. Among these voodoo methods are alternate buffers (apparently removing > potassium salt can improve matters?), different cycling conditions, and > different setup of the reaction. > I gather one of the big limiting factors is mismatches; taq extends > piecemeal, and if it detaches at a mismatch then there's no 3' end bound to > the template for the next enzyme to bind. So any method that enhances enzyme > fidelity should also enhance extension length. > > Does anyone out there have tips on how to reliably achieve up to 4kb > amplification with bog-standard taq? I have a limited ability to make my own > buffers, and I can play with Magnesium and NTP concentrations easily enough. > > *Anyone with information on how much complementarity is enough to induce > recombination in B.subtilis in this case? DNA is generally minced into > single-stranded DNA on uptake, and either end may suffer "nibbling" by > nucleases before circularising stably, so I'm imagining primers with a > 10-15bp 5' "tag" complementary to the other end of the sequence. > > -- > letters.cunningprojects.com > twitter.com/onetruecathalhttp://www.indiebiotech.com
Hi, 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 Hope this helps _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
