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role of MgCl2 and DTT in PCR - (Dec/05/2006 )

Hi ...

can any1 help me with the reason for MgCl2 and DTT being used in the reaction buffer for RT and PCR?..sorry if i may sound amateurish... but i really got to knw..

-rashmi r-

not sure, but i think MgCl2 is used as a cofactor for enzyme activation, and DTT is a reducing agent

-strawberry-

Mg also binds to your DNA, both template, primers and dNTP's. The amount of Mg will influence melting of dsDNA etc, so it influences primer annealing as well.

-vairus-

Mg++ - one of the main variables - change the amount added if the PCR result is poor. Mg++ affects the annealing of the oligo to the template DNA by stabilising the oligo-template interaction, it also stabilises the replication complex of polymerase with template-primer. It can therefore also increases non-specific annealing and produced undesirable PCR products (gives multiple bands in gel). EDTA which chelate Mg++ can change the Mg++ concentration.

DTT is a reducing agent, so it helps to break bonds (like disulfide bonds) which will loosen the secondard structure of the RNA and facilitate RT enzyme initiation of transcription and processivity.

-aztecan princess-