oligo(dt) 15 vs random primers - (May/03/2012 )
I'm using the High Capcity cDNA Reverse Transcription kit from applied biosystem. The kit comes with random primers, however I wanted to try oligo(dt) 15 primers. I was wondering if I would need to change my thermocycler conditions. everything I've seen online are kit specific so I'm not sure what I should do.
The kit (superscript III) that I use says to incubate oligodT primed RNA at 50 deg C for 50 min, but for random hexamers to incubate at 25 deg C for 10 min, then 50 min at 50 deg C.
So it appears there is a minor difference in the conditions, but how this affects the product, I don't know.
We are comparing the High Capacity cDNA kit to our usual Superscript III First Strand Kit. I would like to know how things went with your try of oligos instead of the hexamers. We have always used oligos with the Superscript kit, and I'm hesitant to switch to hexamers in the High Capacity kit. Any comments?
I think Fermentas' cDNA premium kit is one of the best out there. we usually use gene specific primers (gsp) instead of random hexamers or oligo(dT). Our pcr works much better with gsp. we also heat our samples at 65C before addition of buffer and RT enzyme for 5 min. This is in the protocol. Again, pcr works better if we pre-incubate at 65C. After addition of RT we incubate at 50C for 30-45 min. I know superscript is very popular in the US, but I would still go for the premium kit from Fermentas. Thermo just bought them last year, so they must be sold under Thermo now.