Dear all,
I am planning to make IVT with a plasmid I already have.
The plasmid is composed by:
..........T7 promoter - cDNA of my gene - P2A - eGFP - bGHPolyA ............
Looking in the datasheed of NEB IVT kit #E2060S it is recommended to linearize the plasmid exactly at the end of the PolyA site...
Could be an issue if I have a (blunting) restriction site at +29 from the end of the bGH Poly A site? (Otherwise I have to introduce a new restricition site by site directed mutagenesis...)
I think it should be fine. but if you check the T7 IVT document from Invitrogen, which stated that some 5' overhang or 3' overhang will affect the yield of the IVT RNA.
Thank you Junjie,
I think that the nest think to do is to try!
(Even if in my mind the biological mechanism that could affect the yeld if there are some extra bases after the polyA site is not so clear...)
I will try to produce the mRNA and I will check the yield.
May be could be usefull to check the presence of the polyA by a PCR with oligodT at the 3'... I will think about it...
I think bGHPolyA would not contribute PolyA while IVT. bGHPolyA is a ployA signal that needs the protein complex in cells to serve the function. You need to use the enzyme provided with IVT Kit to add polyA.
Dear Xiaoju,
thank you for your answer.
Just yesterday I realized what you said by analyzing the datasheet of a the NEB for IVT.
So, at this point, I am wondering what is the optimal bp lenght after the end of my cDNA to cut/linearize the plasmid. If I leave about 200bp after the stop codon,, could it be fine for the mRNA production and the subsequent polyA tailoring?
I am Xiaoyu but not Xiaoju, haha.
Don't worry about the length of the sequence after the STOP codon. If you want to add some sequence such as 3'UTR to study the biological function of these 3'UTRs, you can add these sequences after the stop codon, and if you don't care about the 3'UTR, you can also add no sequence after the stop codon. After IVT, the enzyme will add polyA after the stop codon. With or without 3'UTR after the stop codon both OK for translation, and I had used both of the sequences to do IVT, and the protein can be translated with both sequences. So, it all depends on your aim. By the way, YOU MUST take attention to the kit you used, to confirm that this kit is suitable for long-template.
Dear Xiaoyu, sorry for the typos 😅
Thank you for your precious informations.
Yes, the NEB kit is fine for long transcripts.
I have a fine restriction site 200bp after the stop codon (I am not interested in the 3' UTR study). In your experience, this restriction site could affect the reaction if it is not blunt-end?
For IVT, I had used templates generated by PCR with Taq enzyme that adds additional A at the end of the 3' terminal. If you worry about annealing between sticky ends, you could apply double enzyme digesting, though I think it is ok with sticky ends.
The following words could be helpful from the Kit I used for IVT.
"Although we routinely use all types of restriction enzymes, there has been one report of low-level transcription from the inappropriate template strand in plasmids cut with restriction enzymes leaving 3' overhanging ends (produced by Kpn I, Pst I, etc.; Schendorn and Mierindorf, 1985)."
"Note that DNA from some miniprep procedures may be contaminated with residual
RNase A. Also, restriction enzymes occasionally introduce RNase or other inhibitors of transcription. When transcription from a template is suboptimal, it is often helpful to treat the template DNA with proteinase K (100–200 μg/mL) and 0.5% SDS for 30 min at 50°C, follow this with phenol/chloroform extraction (using an equal volume) and ethanol precipitation."
"Digest with appropriate restriction enzyme Use an enzyme that will linearize the plasmid so that the polymerase promoter site will be upstream of the sequence you want to transcribe. The volume of the restriction digest should be about 2–3 times the volume of plasmid DNA used. Follow the recommendations of the restriction enzyme supplier for buffer composition, units of the enzyme to use, and incubation conditions."——mMESSAGE mMACHINE® T7 Ultra Kit User Guide
By the way, why not try PCR templates with high fidelity enzymes?
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Biology
- Biomolecules
- Nucleic Acids
- RNA