Whether Mg2+ can be released from the Reverse Transcriptase after Its Inactivati - Urgent (Jun/29/2006 )
Recently I encounter a problem When I do RT-PCR. I add 10ul 2.5M Mg2+ (Recommended by Invitrogen) into RT system. I wonder that whether Mg2+ will be released from the Reverse Transcriptase after Its inactivation? whether the Mg2+ Titration will affect the latter PCR reaction? In other word, Should I consider the Mg2+ Concentration in RT when I Titrate Mg2+ in PCR?
Thanks in advance!
The Invitrogen Superscript protocol I have recommends adding MgCl2 to a final concentration of 5mM per 20uL reaction, or a 1:5 dilution of 25mM stock MgCl2 solution. Using a single uL of the cDNA product would then only add 200uM of extra MgCl2 to the PCR reaction, provided all of it is available to the DNA polymerase. The actual amount may be much less.
That said, it looks as though the amount of MgCl2 you're adding is very high. Double check the Invitrogen protocol, as that may be whats causing the problem.
Depending on how much Mg is in your RT reaction and on how much you need in your PCR, do consider the amount you transfer from RT to PCR...
Hi, Elias and vairus:
Thanks for your replies!
I add the Mg2+ strictly accroding to the Protocol offered with Invitrogen Kit, which I upload in the attached files.(page17), I add 2ul RT product as the Template in PCR Rxn. All You mean that the Mg2+ concentration added in RT should be considered in thge latter PCR Rxns?
BTW, the Final concentration of Mg2+ I added in the RT is 5uM, the total Volumn of the RT is 50ul!
I just can not repeat the RT-PCR, which I have succeeded before. The total RNA I isolated from the Same tissue (different individuals) is Intact identified by Electropherosis. and RT condition is same.
I also wonder if too much total RNA (more than 5ug) input in the RT rxns will inhibit RT rxns?!