PCR 10x Taq Buffer with MgCl2
wojtek777 at poczta.fm
Tue Oct 3 03:07:38 EST 2006
Dnia Fri, 15 Sep 2006 17:22:09 -0400, aria johnson napisał(a):
> Hi Alex,
> The Mg ++ neutralizes the replusion between the primer backbone and
> template DNA backbone and this affects primer binding stringency. The more
> Mg you have the more it neutralizes the repulsion / the better the primer
> can bind to the template strand - the less Mg is the more the repulsion
> between the primer and template resulting in less primer binding to the
> template. As a result, too much MgCl can lead to a lot of non-specific
> amplification (the primer can bind to sites with mismatches) while too
> little MgCl can lead to no amplification (repulsive forces too much).
> Mg is also a cofactor in DNA polymerization - the more Mg is the better
> the Taq is able to perform but here again - too much Mg can reduce the Taq
> enzyme fidelity while too little can can inactivate the Taq.
> As you have asked about components of PCR buffer you should note, MgCl
> does not have to be in the 10x Buffer - we use different concn of MgCl in
> different reactions and so buy our 10x PCR buffer separate from our MgCl.
> take care
> ps there is a good pcr book called PCR Primer: A Laboratory Manual by Cha
> and Thilly (1995) that can be helpful and also Molecular Systematics that
> can be helpful to you. aj
> On 9/14/06, Alexander Berkow <alexbrkw at gmail.com > wrote:
>> Can someone out there tell me what the purpose of the MgCl2 in the 10x
>> Taq PCR reaction buffer is and what else is in the buffer that is
>> necessary for
>> the PCR to work correctly?
>> _______________________________________________ Methods mailing list
>> Methods at net.bio.net
> Aria Johnson
> Biological Scientist,
> 7922 NW 71st Street,
> University of Florida
> Gainesville Fl. 32653
> U.S.A .
One more remark. dNTPs bind Mg2+ ions in stechiometric manner (mole of
Mg2+ per mole of dNTPs). The concentration of magnesium ions must be
optimal for Taq polymerase ABOVE the dNTPs concentration. I checked this
phenomenon personally and it seems to work that way.
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