HELP! In-vitro transcription results only in smear! - (Feb/09/2007 )
I'm really frustrated and really need help. I'm trying to establish in-vitro transcpription in a new lab since months and the only thing I get is a smear so here the details.
I have clone my gene of interest in a vector containing T7 Promoter sequence. I designed Primers upstream including T7 promotor and downstream of Poly(A)signal in order to be able to use the primers for each construct with this vector.
1.For PCR I use the plasmid derived from a Qiagen maxiprep diluted to 8ng/µL. (1:1000 diluted). I perform PCR with Promegas GoTaq and RNase free water.
2.After PCR reaction I added 20µg Proteinase K (Roche) to a 50µL PCR sample and incubated for 30 min at 50°C. (Final concentration is 400 mg/mL) And then 20 min at 70°C to inactivate Proteinase K.
3. Meanwhile I run an electrophoresis to check my amplicon which is always ok.
4. After treatment with PRoteinase K I clean up my samples with Promegas Wizard PCR GEl extraction clean-up Kit. I do not perform a gel extraction, but follow the clean up procedure. I eluate the DNA with Nuclease free water included in the kit.
5. I use the sample and do the in-vitro transcription with Ambions mMessage mMAchine T7 Ultra Kit. Following the protocol including treatment with DNAse I TURBO and Poly A tailing.
6. Then I clean up the RNA using Qiagens RNeasy Kit and eluate the RNA in the included RNase free water.
7. When I run a denaturing formaldehyde electrophoresis the only thing I can see is a smear starting at the size where my transcript should be.
I repeated this procedure and it is always the same. The first time my RNA Marker was also degraded so I thought this was due to contamination. So here the list with the things I changed after supposing contamination.
1. I have a special pipette set only for RNA which I wipe before use with RNase away solution.
2. I only use prefilled certified pipette tips. (I even used a complete new set of boxes just opened by me)
3. I just opend a new bag of eppendorfs which I use directly from the bag.
4. Of course I always wear gloves. I really pay attantion to change them often in between working.
5. First in-vitro transcpriptions were performed with Promega RiboMag large scale kit. Since I assumed it was contaminated I bought a new one from Ambion. But even with this complete new kit nothing changed.
6. Also the PCR clean up Kit is complete new.
7. I even bought a new Qiagen RNEasy Kit, also used the first time by me. (I think the kit is ok, since total RNA Isolation performed with the same reagents looks good on the gel)
7. I prepared complete new MOPS buffer. The loading dye is bought from Fermentas. ALso complete new vial. (Since the first time my RNA ladder was also degraded I thought contamination maybe ocurred during electrophoresis.) But now the RNA ladder is perfect. No smear!
Electrophoresis works well I mix buffer and add after 30 min some additional fresh one. Since my ladder looks good now I can be sure that only my sample is contaminated.
I hope it is not too much information but I really tried everything and do not know what else to do. I may be new to in-vitro transcription but I am experienced in dealing with RNA since until now I performed lots of expression studies doing northern blots, rtPCR, microarrays etc. NEver had problems with degraded RNA!
I think now it must be the DNA template but I treat it after PCR with Proteinase K. The only thing I didn't do is to change PRoteinase K. I didn't buy the PCR grade one frome Roche but used the Proteinase K included in Roches gDNA Isolation high purity PCR template Kit. So I assume the provide the PCR grade PRoteinase K. The stock is in -20°C from september 2006. I thaw it and then store it up to two months in 4°C.
So I am open to any suggestions I don't know what else to do. I really need help otherwise I'll get crazy.....
I'm not sure whether you have already solved this problem, as your post is a little old, but thought I'd post a reply anyway. I used to have a similar problem to you - I used a method very similar to yours, and always ended up with a big smear of trancript. I was then told by a hardened molecular biologist to skip the DNase step in the Ambion protocol, and precipitate with LiCl, as Li selectievly precipitates RNA. I was a little wary at first - but ended up with perfect message, which not only looked pretty on a gel, but behaved perfectly biologically.
If you are still having problems, it might be worth giving it a shot!
did you try with linearized plasmid?
second, i would try 37°C proteinase K treatment, and then doesn't inactivate at 70°. I think RNA is not very stable at 50° and less at 70°. In total your RNA is 50' !!!!! at 50° or higher. dennaturation step in my transcriptions in vitro is 2' at 95, but may be done 5' at 56°
Second, i treat my transcriptions at 37° with the proteinase K.