I then used Invitrogen's double-stranded cDNA synthesis kit ..
Protocol for NEBNext® Ultra™ II Non-Directional RNA …
I'm generating cDNA from purified poly(A)RNA, following a protocol and noticed that there is no second strand primer. AND, the thermocycling conditions for the second strand are fairly cold - 2hrs @ 16C. Is it possible to have cDNA synthesis occur spontaneously without a primer? I'm wondering if this is a missing element in my protocol; the first strand primer (a polyT primer) shouldn't match the first strand synthesized, so what starts the second strand??? And what does the DTT do?
Thanks for any info!
Here's the scheme - most reagents are supplied by Invitrogen:
First Strand Synth -
dT (polyT) primer
first strand buffer
DTT (what the heck is that?? comes with the superscript kit)
SuperScript III RT
Second Strand Synth
second strand buffer
T4 DNA polymerase
EDTA to stop rxn.
how can we design primer to synthesis second strand of ..
ProtoScript II – NEB ProtoScript II First Strand cDNA Synthesis Kit and random primer mix; SuperScript II – Invitrogen SuperScript First Strand Synthesis System for RT-PCR and random hexamers; SuperScript® III – Invitrogen SuperScript III First Strand Synthesis SuperMix and random hexamers. The following reagents are supplied with this product: