I'm trying to get in vitro transcribed RNA from a linearized plasmid, using the T7 MEGAscript kit. After the reaction I run a few ul on a 1% gel to check my RNA, but sometimes I get no RNA product only a faint band that runs at the same height as my linearized plasmid DNA (control), which is gone after I do a DNAse treatment. I take the kit control along, so the reaction set-up is okay, but I can't figure out why I don't get RNA from my linearized plasmid DNA. Does anyone have some tips? I follow the protocol as described in the manual from Life Technologies.
T7 is fairly sensitive to salt concentrations, so it could be that your DNA template is not sufficiently purified to use as a template. How are you preparing your template and how much DNA are you adding to the IVT reaction? Alternatively, maybe this plasmid isn't what you expect and lacks the T7 promoter.
Have you sequenced your vector to make sure it is what you expect?
RNase contamination could be another culprit. If your positive control from the Megascript kit is working, try spiking in a bit of your template of interest into the reaction. If this fouls up the positive control, then you know there is a salt or RNase contamination issue with your template. If it doesn't, then it is likely that your template doesn't have a functional T7 promoter, or your restriction digest cuts the vector between the promoter and cDNA (perhaps your restriction map is wrong?).
Thanks for all the tips. I'm pretty sure there's a T7 promoter and it's not removed or destroyed when linearizing the plasmid (plasmids also have been sequenced by a colleague). With the same source material, I sometimes get RNA and sometimes I get no product. RNAse contamination seems likely, the only source I can think of is in my plasmid DNA :( I already use dedicated 'RNA only' plastics.
Briefly, this is how I prepare the linearized plasmid:
restriction digest of ~8 ug DNA, followed by gel purification using a silica based method (it's something they use in this lab and as I started this new job recently, I'm not sure on which protocol it is based, but everyone uses it). I can see one problem with older samples, as the protocol describes to elute using TE. From molecular cloning, I know TE can interfere with downstream processes.
For the T7 Megascript kit I follow the protocol and my input is 0,5-2 ug (I mean I've tried a range already between 0.5 and 2 ug. Last week I tried 2 ug and it worked, but before I used the same sample with 1 ug and was also fine, though the next time... not)
I incubate 3 hours at 37C. I can see another problem here, but I'm not sure how much of an issue this is: temperature. I checked one well in one heatblock and the temperature was off by 1 degree C, but I don't know if this is the case for all wells. I've now switched to using a waterbath that I checked at 37C, but yeah I don't know how temperature sensitive it is.
I also want to do the check they suggest in the manual for inhibitors or possible RNAses (though I've also found the enzyme mix contains an RNAse inhibitor)
Agreed that the problem is likely RNAse contamination in your template DNA. In terms of RNase inhibitors, I've had good success with the murine RNase inhibitor from NEB. You should try to use RNase-free water for your plasmid DNA instead of TE. Since water does not elute DNA as efficiently in the silica purification systems, you might opt for ethanol precipitation instead.
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