RNA in vitro transcription - kits, protocols - recommendations? (Jul/13/2005 )
Does anyone of you have experience or recommendations for in vitro RNA transcription systems, such as Riboprobe (promega) or MaxiScript (Ambion)?
What about alternative protocols? I have cloned my template into pGEM, could I used buy a RNA polymerase and do it myself?
How about the RNA yield (the RNA shall be used for RT-PCR as a standard).
Thanks for any input!
I use Roche products to make RNA with Dig UTP label. You can buy the kit or buy the components separetly.
I have also used promega products. The T7 RNA polymerase comes with buffer and DTT. you can get the NTP's and Rnase inhibitor for the synthesis of non labeled RNA. The protocol is straight forward. make sure you linearize the plasmid and avoid using enzymes that create 3'overhangs.
The yield should be enough for RT-PCR. Probably have to DNase and precipitate RNA before using it.
I've tried both Roche and Ambion (mMessage mMachine) kit for T7 and Sp6 transcription. The kit from Roche is cheaper, but Ambions kit gave a much higher yield.
I recommend the kit from ambion also... works well; those boys in Texas know what they are doing.
I used the ambion, sufficient yeild, great stuff
Ambion Maxiscript is teh gr8tness.
high yield, EASY TO FOLLOW INSTRUCTIONS (yay), great results. You could try to do without the kit, but the results wont be as good.