role of MgCl2 and DTT in PCR - (Dec/05/2006 )
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..
not sure, but i think MgCl2 is used as a cofactor for enzyme activation, and DTT is a reducing agent
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.
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.