After a long break and a lovable toddler I am back in science - I run my own project
Right now I am trying to pick up my primer pairs for RT real time-PCR and I have a dilemma:
In the past I was always performing the RT-reaction on the bench and then proceeding to the real time-PCR.
The primers I designed were for products 100-120 bp long, for reasons of economy the volume of the reactions was app. 12 microliters - I never had problems.
I wonder now, if I increase the volume of the reaction up to 25 microliters (this is the original protocol by the manufacturer of the master mix) could I amplify a bit longer fragments, 150 bp for example?
I know that the recommended length is 80-120 bp, but does anybody has an experience with longer fragments in larger volume of reaction?