How to improve DNase efficiency after in vitro transcription? - (Jul/23/2008 )
Hi,
After in vitro transcription to get ~8kb RNAs (using MEGAscript Kit from Ambion), I treated 19µl of the transcription product with 1µl of RNase free DNase 1 (2U/µl), 15 minutes at 37°C.
Then I purified the RNase with MEGAclear Kit from Ambion.
PCR + gel showed there was still DNA, so I re-treated 99µl of "purified" RNA with 1µl of DNase, again 15 minutes at 37°C.
qPCR showed it wasn't enough to get rid of all DNA.
Does anyone know how to improve the efficiency of the DNase treatment, by using a buffer or greater quantities of DNase?
Thanks a lot
Hi soam,
be sure that your buffer contains MgCl2 and CaCl2 and is in the range of pH 7.0-8.0.
I can recommend you a recombinant DNase I which is not purified from bovine pancreas but from a non-animal organism.
Native DNase I is ordinarily prepared from bovine pancreas, one of the richest sources of RNase activity.
Maybe one of your collegues has the DNase I recombinant, RNase-free (Cat. 04716728001) from roche in his freezer and you can borrow some of the enzyme and buffer. I think they only have a relatively big pack sizes which would be enough for you for years if you hardly need it. Maybe you check out their website or look if there are also other suppliers which offer this quality of dnase...
be sure that your buffer contains MgCl2 and CaCl2 and is in the range of pH 7.0-8.0.
I can recommend you a recombinant DNase I which is not purified from bovine pancreas but from a non-animal organism.
Native DNase I is ordinarily prepared from bovine pancreas, one of the richest sources of RNase activity.
Maybe one of your collegues has the DNase I recombinant, RNase-free (Cat. 04716728001) from roche in his freezer and you can borrow some of the enzyme and buffer. I think they only have a relatively big pack sizes which would be enough for you for years if you hardly need it. Maybe you check out their website or look if there are also other suppliers which offer this quality of dnase...
Hi THE PROFESSOR,
Thank you for answering.
The DNase I use is already supposed to be RNase free. And I actually have no real problem with the RNAs.
I'd read that MgCl2 and CaCl2 concentrations were important, but I just add the DNase to my transcription products, which contain the buffer I used for the transcription. Unfortunately, the composition of the buffer is not thoroughly detailed in the handbook of the kit, and the handbook does not mention any particular buffer to use with the DNase.
I think I'll try with other DNases, I know we have some at the lab.
Thanks anyways for your help!
Soam
I doubt your in vitro transcription reaction buffer has CaCl2. You could add CaCl2 to it just prior to DNase I to 1 mM final concentration. That will help DNA digestion.