Plasmid template preparation for in vitro transcription - (Jul/18/2005 )
Hi there,
I am going to transcripe RNA in-vitro from a plasmid DNA. I was wondering whether the plamid I had previously extracted using a kit is clean enough for the transcription reaction.
Any thoughts?
I am going to transcripe RNA in-vitro from a plasmid DNA. I was wondering whether the plamid I had previously extracted using a kit is clean enough for the transcription reaction.
Any thoughts?
I just finished a run-off transcription reaction today (actually staining the gel right now to check transcript size). What kit did you use? Is this run-off transcription? If so, you're going to want to clean up your template and remove the restriction enzymes and other unwanted stuff still in there (PCI/salt/ethanol steps)
Did the kit have RNase in it? If so then it is very likely that your plasmid will be contaminated with RNAse.
Hi,
I used the NucleoSpin kit (BD Bioscience) to extract the plasmid and the first solution during plasmid extraction contains RNase A.
Can I get rid of potential RNase contamination by a ethanol precipitation after linearizing the plasmid?
Freiberger
I used the NucleoSpin kit (BD Bioscience) to extract the plasmid and the first solution during plasmid extraction contains RNase A.
Can I get rid of potential RNase contamination by a ethanol precipitation after linearizing the plasmid?
Freiberger
Not really as RNase "sticks" to DNA pretty well. You will need to use a clean-up method that will get rid of it. Try a few phenol extractions with 0.1% SDS present and then a EtOH precip.
Hi, it's me again
I just got another question (hope it doesn't sound too stupid): do you remove unincorperated nucleotides after transcription?
How do you quantitate the obtained RNA? OD260/280? Isn't that a waste of RNA if the total volume of purified RNA is ~ 20ul?
Thanks for your reply!
Freiberger
What are you using your RNA for? Is it labelled in any way (P32)?
To remove the nucleotides you will need to use a size exclusion spin column or micro ultrafiltration. This is pretty simple and quick.
Hi Daniel,
The RNA is for RT-PCR and not labeled.
Freiberger
Just run 1ul on an agarose gel to check. If you are using a linearised plasmid then the RNA should all be around the same length.
I always treat my plasmid with proteinase K and RNASE followed by Phen:Chlor
after kit purification. Bacterial proteins stick to the plamid. For sensitive in vitro transcription reactions with reconstituted components, this step is very impt.