Help with DIG UTP Pobe Construction In Vitro Transcription - (Jul/05/2008 )
My lab is attempting to perform in vitro transcription using DIG UTP labeling. However, our yields for RNA are very low and we are having difficulty figuring out why. I was hoping that I might get some help if I posted our protocol.
1. Restriction enzyme digest for 3 hours.
2. Phenol Chloroform cleanup followed by overnight ethanol precipitation.
3. Re-suspended pellets in 30 microliters of TE and ran a gel. Gel intensity and band sizes looked great. Decided to move to probe construction
1. Transcription (Set-up at room temp)
2 nanograms of DNA , 2 microliters of 10 X RNA Polymerase Buffer, 2 microliters of DTT, 0.5 microliters of RNAsin, 1.8 microliters of T7 RNA polymerase, 2 microliters each of ATP, GTP and CTP, 1.3 microliters of UTP, 0.7 microliters DIG UTP, filled to 20 microliters with PCR water
2. Allowed transcription to run for 2 hours in 37 degree water bath
3. @ conclusion of 2 hr, added 1 microliter of DNAse and placed in 37 deg. water bath for 15 minutes.
4. @ conclusion of 15 min, added 1 microliter of 0.5 M EDTA
5. Added 2 microliters of 3 M NaAc and 40 microliters of 100% EtOH, placed in -20 freezer for overnight precipitation.
Part 3 Diagnostic Gel
*Digest gel looks fine
* Prednase shows weak DNA bands, strong RNA bands
* Postdnase shows strong RNA bands
* Probe has weak bands
All ran on 1% agarose gels made without EtBr, loaded with RNA Loading buffer and soaked in EtBr solution after being run. Then imaged.
PLEASE HELP ME!!!
I have only experienced PCR labelling with DIG, but already there... it works at once, another time it doesn't... and Invitro transcription is more tricky than PCR... My only guess is that the polymerases generally have a hard time incorporating DIG-dUTP... you could try to lower the amount of DIG label you use in the reaction, others have find it to work (make a search with DIG as keyword in the forums).