In vitro transcription question
Posted 26 March 2012 - 07:12 AM
Is it necessary to do a phenol/chloroform extraction prior to in vitro transcription?
I linearized my plasmid with the appropriate restriction enzyme, and then i purified the plasmid using a zymo DNA clean & concentrator spin column.
Will the DNA from the spin column be pure enough for in vitro transcription?
Posted 04 April 2012 - 06:20 AM
Posted 11 April 2012 - 10:04 AM
I am using Ambion mMessage mMachine kit for in vitro transcription, and I have obtained very good gel results all the time. I directly use linearized plasmids and face no problem. If you obtained good plasmid isolation results and linearized them properly, I don't think you need to take any further steps
You dont purify the DNA after the restriction digest (to linearize)? I lose a lot of yield when I use the Zymo spin column. I am also getting a smear when I run the reaction on a denaturing gel. The manual suggests treating the DNA with proteinase K. Has anybody had to do this before?
Posted 12 April 2012 - 03:02 AM
Posted 12 April 2012 - 07:56 AM
Posted 13 April 2012 - 03:56 AM