Which in vitro transcription kit is better? - (Aug/15/2006 )
Promega or Ambion?
Which company is adepter in in vitro transcription?
I've not used thee Ambion kits but the Promega one is very quick and easy (if you mean T7 RNA polymerase - as in your other posts).
You get a 5x reaction buffer and the T7 RNA Pol.
All you need is a microgram of DNA template, the buffer, NTPs (including a radiolabelled one) and the enzyme. Incubate at 37 deg C for 90 minutes an then add formamide loading dye to stop the reaction. Heat to 90 deg C for 4 minutes, cool on ice and then run the products out on an 8% denaturing polyacrylamide gel at high voltage.
It's really quite simple.
The sequencing lanes aren't so easy though.
We use supplies from NEB for invitro transcription.
We use the Ambion kit. For miRNA transcription, you should be able to get 3-4 ug of RNA per ul if you let the reaction run overnight (16 hours). I like to add an RNase inhibitor so my product is not degraded.