RT-PCR: Two-Step Protocol - MIT OpenCourseWare
The two-step protocol is usually more sensitive than the one-step method; yields of rare targets may be improved by using the two-step procedure. ... If you are testing n sets of primers, make a master mix enough for n+1 tests. Mix the components gently but thoroughly. Aliquot 22.5µl of your master mix to each tube.
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