skip 65 degrees protocol of Reverse transcription - (Jul/10/2013 )
what will happen if i skip the 65 degrees incubation of my RNA and simply throw all components of my RT-PCR mixture with the MML-V enzyme in my reaction tube and allow it to react at 37 degrees for 1 hour and inactivate the enzyme at 85 degrees?
The first step at 65 or 70 degree is for denaturing RNA secondary structure so that the primer can bind to the RNA when you subsequently chill the reaction on ice. This step is optional but important for long messenger RNAs and GC-rich RNA regions and can increase cDNA yield.
thank you, if i am going to generate my cdna for qPCR using taqman system, and i designed the primer with intron spanning, do you think, i need not worry that much if i skip this denaturing step? thank you ...
You should be OK. BTW, which type of primer did you use for the RT reaction?
i used oligo dt... should it be fine? if i use 2 ug of rna template and .5 ug/ul oligo-dt in a 50 ul reaction tube, and used 20 ng of cDNA template to my qPCR, should it be fine?