Long-amplicons and Taq

chovek69 via methods%40net.bio.net (by ivanoov from gmail.com)
Thu Jul 7 06:47:25 EST 2011


On Jul 6, 3:26 pm, Cathal Garvey <cathalgar... from gmail.com> 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


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