MMLVRT Temperature - (Jun/02/2005 )
for a RT, I have a programm already make by somebody in the labo but is somebody say me if my idea over the temperature is correct?
10 minutes at 70°: for the annealing of the pd(N)6 to RNA
45 minutes at 42°: for polymerization. Is it the optimal temperature of the MMLVRT?
3 minutes at 99 °C: for the dehybridyzation between the new products?
To be more precise:
Firstly, you should incubate your RNA and primer at 70oC for 10 min, this is to denature all the secondary structure of you RNA template. After that, chill imediately on ice. This will allow your primer annealon you RNA template. Over all this step is to provide batter specificity of your RT step.
Secondary, add your RT buffer and RTase into your sample + primer solution (make up to the right concentration).
Start the RT reaction by incubate at 42oC for 45min.
Finally, incubate at 99oC is to stop the RTase from reaction.
Thank you Hadrian!!!