Hello everyone!
I am currently working on a project that involves the use of mRNA. I started off my ordering plasmid from VectorBuilder (which contains T7 Promoter, gene-of-interest, Poly-A-tail sequence and all other necessary genes for bacterial selection -> I've attached the diagram of the plasmid as well.) The plasmid contain site for both BspQI and SapI at the very end of the Poly-A-Tail section of the plasmid.
Currently, I have finished amplification (through E. coli) and linearization of our plasmid (using BasQI restriction enzyme) already. I have verified that the plasmid has indeed been linearized using gel electrophoresis which I have included in the picture below (well#5 is the linearized plasmid while well#3 is the same uncut circular plasmid for comparison). I am now working on translating the linearized plasmid into mRNA using the T7 MEGAscript Transcription Kit from Thermofisher. After the mRNA was synthesized, I used the MEGAclear mRNA purification kit to purify the mRNA. I tried my best to adhere as tightly as possible to the given protocol. However, the nanodrop (spectrophotometer) showed almost no mRNA yield at all compared to the control which was given by the kit (the control is also linearized plasmid).
Do you have any idea of what might be the cause of the problem? Or some condition that might have to be altered? I had tried my best to follow the protocol strictly but maybe I could have missed something?
Thank you so much!
Settanan Plangsiri
P.S. For further information, I have included the picture for my nanodrop down below! (The normal one is the control while the one with super weird graph, error signs and low yield is the gene that I am trying to transcript and use for my project!)
it is hard to say what the problem is, but assuming that I am counting lanes correctly the amount of linearized plasmid you have is very low. I would guess that you have maybe 10ng loaded on that lane. So it is possible the issue is merely insufficient template, I am not sure how much you need for the transcription reaction.
Your mRNA might be too long to be efficiently eluted from the MEGAclear cartridges under conditions specified in the kit's manual. You may try to elute it by several consecutive 50-mcl portions of water. Also, you may let the water stay in the cartridge for 5-10 min before centrifugation to increase contact time with the sorbent. Using weak EDTA solution instead of water may help too (something like 1 mM EDTA, pH 8).
Actually, it might be useful to set up the T7 transcription reaction in small scale (~ 20-30 mcl), and then resolve the reaction mix on a gel to see if you had the mRNA synthesized in the first place.
Promega Note "T7 RiboMAX™ Express: Generation of 27kb in vitro Transcripts in Minutes" describes an alternative way to separate the synthesized mRNA from other components of the reaction mix. After digesting the template with DNase, the mRNA is precipitated with LiCl and collected by centrifugation:
"... After adding 1U of RQ1 RNase-free DNase to the reaction and incubating at 37°C for 15 minutes, the RNA was precipitated by adding 30µl of nuclease-free water and 25µl of LiCl solution (7.5M lithium chloride, 50µM EDTA). The reaction was chilled for at least 30 minutes at –20°C and centrifuged (maximum speed in a microcentrifuge at 4°C for 15 minutes). The resulting RNA pellet was washed with 70% ethanol, dried and resuspended in 1mM sodium citrate (pH 6.4) ..."
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