Hi everyone, I am working on in-vitro transcription to generate mRNA for LNP fabrication. The kit I am using is the HiScribe® T7 mRNA Kit with CleanCap® Reagent AG from NEB. The first time I was doing it in October, the yield was very high. But the yield has been getting lower and lower since the second time.
Even with the control template the kit provided, the yield is always lower than 7 µg/20µL reaction.
Then, the technical support sent me a new kit, but the control template generated an even lower yield.
I've been talking to the technical support for a whole month. I am sure that I always put the T7 polymerase mix on ice, add reagents in the same order as the protocol, and set the reaction in room temperature. I am always aware of the RNAse. There was no RNA degeneration diffuse under my RNA band. The electrophoresis always shows very fainted bands even from the aliquot of unpurified 20µL IVT reaction.
Now, I suspect that the enzyme just started degenerating since I opened the T7 polymerase tube. Does anyone encountered this situation before?
A few questions:
1. How long do you let your reactions go for?
2. Are you adding DTT to the reactions?
3. Did you run the RNA on a gel to see the quality and the products you are making?
4. How are you storing the reagents? T7 RNAP should not go bad at -20C that quickly. I have used the same enzyme from NEB for many years without any issue.
Thank you so much for your reply!
1. I have tried 1h to 3h and overnight reaction. The first time with the high yield is using 2h.
2. I added DTT every time (1µL per reaction) except the first time because there was some precipitation in the DTT tube.
3. I ran the RNA on the gel directly from an aliquot from the IVT reaction. In the attachment uses the control template from the kit. In the picture, the band in the red box represents the right product of the CLuc template. But the band is so faint that I have to increase the contrast in Image Lab. In the bottom of the picture, there is a diffusion but it is not connected to the expected band so the technician didn't think it is degenerated mRNA. (1☓MOPS, 2% denatured agarose gel, 100V, SYBR GOLD stained).
4. T7 RNAP was stored at -20. When I am using the T7 RNAP, I put it on ice except I pipetting from the tube.
Thank you again for your advice and experience. I am happy to see that you are get along well with the NEB enzyme. May I ask if you always aliquot the enzyme into small portions to avoid repeated freeze-thaw cycles? How long can the activity of enzymes generally be maintained?
Hmm, might be time to double check settings on any shared equipment. I've had folks edit my saved and named programs in PCR machines (eg. "65 degree hold" was changed to a different temperature). The enzyme should be fine. Some of the reagents (DTT, RNA bases) can degrade more quickly. Are you the only person using the kit?
Yes, especially the DTT. It can also precipitate then you won’t get the right amount
are you using filter tips? Can you clean your pipets?
Thank you again for your help! I hope my experience can help. did it by improving the quality of the plasmids. I was using the phenol-chloroform method as described in the manual to purify it after linearization, which has a process of freezing the purified plasmid with NaAC and Ethanol for precipitation. It was time-consuming and harmful to my plasmid. Because the plasmid template could somehow inactivate during the freeze-thaw cycle. I finally used the purification kit, which is easier and allows me to use fresh plasmid every time. I am kind of sure thatan this freeze-thaw cycle damaged the plasmid (or maybe by other mechanisms) because once I freeze the plasmid, it won't work for IVT again.
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