in-vitro transcription - (Jun/09/2005 )

Hi,

I am using Ambion's t7 in-vitro transcription kit. First, I linearize my plasmid with an enzyme that cuts right after the gene. Then, i dephosphorylate, and extract with phenol:chloroform. I use 1-2 ug for the transcription reaction and follow the protocol given by the company exactly. When I read the concentration in the spectrophotometer, I get a pretty good yield (about 20 ug total).

The problem is that when I run the gel, I see a band but it is very smeared! I am really really really careful about RNase contamination but I still get a smear instead of a sharp band. The other problem is, sometimes, the size of the RNA band is small than it should be even when I am using a plasmid that has worked before.

Does anyone have any ideas?

Thanks!

Hi

Is the band truly smeared or is it more of a ladder?

A ladder is common and is due to premature termination of transcription. If this is the case, try runing the reaction at a lower temperature, like 30C.

Bye.

Dear Roshni,
What RE that you use to lineralised you your plasmid? Did you chose the correct RE buffer?
Because incorrect buffer condition can promote unspecific cutting.

Chech this out please. ok.

Best reards
Hadrian

Hi,

I just run across this posting. I am having trouble with my in vitro transcription too. I use a PCR product fused to the T7 promotor site (~260 bp). The amount of template is about 1 ug/ in vitro transcription. After running the product on a gel, I got 2 bands - one which corresponds to the right transcript size, one that is about twice as big. The last time I even got another smaller one too.

I run the PCR on a gel prior in vitro transcription. One time, I used a PCR cleanup kit the other time I gel purified the PCR product...

Does anyone has an idea what might be wrong in the reaction? Could I try to gel purify the correct RNA transcript?

Thanks a lot!
Freiberger

Hi,

I recently performed the same reaction and was having similar problems. It seems like unincorporated NTPs were not being completely removed after PCI extraction and purification of the transcript. This caused my spec readings to be off and subsequently I would load significantly less RNA on a gel than I should have.


Anyways, make sure you run either RNA markers, or a sample of RNA that you know is not degraded as a control. This will tell you if your sample is messy, or if maybe your buffer went bad during electrophoresis (I'm assuming that you periodically mixed the buffer during the run).