Long-amplicons and Taq

Jonas Danielson via methods%40net.bio.net (by Jonas.Danielson from biochemistry.lu.se)
Fri Jul 8 03:54:44 EST 2011


Hi!

I don't think your main problem will be the length of the amplicon, but rather that you are extremely likely to get errors, on average a 4kb product, when run for 20 cycles will have 2 errors, and finding a clone with no errors will be almost impossible. If this is not a problem for you, fine go on with Taq, but otherwise, change to something like Phusion because the extra money you will spend on the enzyme you will easily save on not having to sequence that many clones (and it will save you some time too).

/jonas

On Jul 7, 2011, at 13:47 PM, chovek69 wrote:

> 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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