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In vitro Transcription - (Jun/04/2007 )

Hi all,
So Im trying to do Invitro transcription and I have 2 problems:

1)cant get rid of the DNA template despite using RQ1Rnase-free DNase
2)How do i know exactly how much RNA i have in my sample if my DNA template is 1ug

Anyone experienced in Invitro procedures, Please help!


well after doing in vitro transcriptions, why do you do a DNase treatment? for which purposes?
i think something in your mix should inhibit your DNase ? What is in your mix? how much salt do you add?
you may also buy a fresh DNase. I prefer turbo DNase... but that's only my opinion.

Second : after you did your DNase treatment, you need to filter your prep in a G25 column to remove all nucleotides that interfers with OD measurements.
then you need to quantitate your RNA by OD and check how many products you have on a gel to estimate how much you have.

I would go for an agilent techology which allow to do that in almost one step.