Please let me know the best starting material/strategy for in vitro transcription process out of the following.
1- Circular plasmid (insert in TA cloning vector) followed by PCR with dsDNA primers
2- Double digestion of Circular plasmid and release the insert from the vector, gel extraction and PCR with dsDNA primers
3- Single digest of circular plasmid, PCR with dsDNA primers.
Hi Bharat,
One question, in none of these do you mention about performing a transcription, only a PCR.
If it is indeed an in vitro transcription you would like to do, do not use circular plasmid as a template as the RNA polymerases are highly processive and will transcribe the entire plasmid.
You have two options:
1) Linearise the plasmid at the end of the sequence you would like to transcribe, purify the cut product and then do the transcription.
2) Design primers, one of which has the transcription promoter as a 'tail' adjacent to the sequence you are targetting (make sure the promoter is on the correct primer), the other as a standard PCR primer. Perform a PCR, purify the PCR product and then use this as template for your transcription.
In my experience we always got better efficiencies when transcribing from a plasmid as opposed to a PCR product.
Hope this helps.
Hello Mike,
Nice to hear from you. I have conducted my first lot of experiments, wherein i have conducted PCR with dsDNA primers (in this case one DNA template with opposing T7 promoters at 5' ends of each strand and used in a single transcription reaction) using the circular plasmid as original template. I have got around 1.6 micrograms/microlitre of dsRNA after purification. Alternatively, i have been double digesting my plasmid and gel extracting and purifying my insert, followed by a PCR with dsDNA primers and purification of the same for the conduct of in-vitro transcription reaction. Do double digests work well for in-vitro transcription process.
I'm not sure I am following you. Does the plasmid itself have transcription promoters or are these added by the primer during the PCR?
Dear Mike,
The construct (TA cloning vector and my insert) is my original template. The dsDNA primers synthesized contain the opposing T7 promoters. It means that the transcription promoters are added by the primers during the PCR reaction.
OK, so you have to perform the PCR for the transcription. That being the case I don't imagine there would be too much difference in what you use to generate your PCR product. If it were me I would do it from the whole plasmid as it is less labour intensive and I can't think of a reason why a PCR from a cut fragment would be more efficient than from a whole plasmid, although I could be wrong. However, it seems your yields are reasonable anyway...
It seems you have performed it by both methods anyway, how did the yields compare?
Dear Mike, Nice to hear from you. Though the dsDNA values were good with the cut fragment, the dsRNA values were lower after in-vitro transcription process. Can you please let me know the procedure of linearizing the plasmid and purification without the need for PCR. I would like to work with the method.
I'm not sure if it would work with what you need to do, I was thinking about only generating ssRNA transcript. This is simple from a plasmid as you just insert a polymerase promoter 5' of what you want to transcribe, linearise the plasmid at the 3' end of the sequence to be transcribed, purify the cut DNA and transcribe as normal.
I guess you could clone a T7 (or T3/SP6) promoter either side of your sequence of interest (making sure the sense is correct for each strand), cut at either end and transcribe from that. However, it is not something I have ever done so I have no idea how well it would work...
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