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in vitro transcription with T7 promoter - (Aug/14/2006 )

Hi,
I am relatively new to molecular and I am in the process of setting up a real-time assay and require cRNA/mRNA to form a standard curve.
My desired fragment is in a TOPO pCR 2.1 vector (invitrogen) which has only got a T7 promoter site downstream of my insert. My insert is also lying 3' to 5' in my vector. I am under the impression that I require the sense strand cRNA for my real-time primers.

My question is how do I get sense strand when my T7 promoter is downstream?

I have come across technique also of using PCR to attach promoter sequence to PCR product with promoter/gene specific primer, can this be down using plasmid as template? And how does this work? I have also come across this in kit form.

So really I was hoping someone could tell me the easiest way to obtain sense strand cRNA/mRNA when the T7 promoter is downstream.

Thanks

-mbarney-

Don't think you can.

The simplest way is to order new primers containing a 5' T7 promoter and just PCR from the plasmid. This will work absolutely fine. Then you'll have a short linear fragment with a correctly orientated promoter and sequence.

Either that or you can reclone but this will take time.

I've used the primer technique dozens of time for IVT studies. See the following for an example:

http://www.ncbi.nlm.nih.gov/entrez/query.f...l=pubmed_docsum

Look in the IVT and methods sections.

I reckon from right now [depending on how quick your oligo supplier is] it will take less than two weeks to do the whole thing.

-Doc_Martin-

Thanks for that.

I will take the promoter PCR approach, but still confused about the orientation of my insert in the vector...
If my insert is lying 3'-5' then do I design the promoter sequence and gene specific primer to read 5'-3' or as the insert lies 3'-5'.

Also if my insert is lying 3'-5' is that technically the antisense strand meaning that if I carried out IVT on my plasmid as is i would produce a strand that reads
(Insert 5'-3')-T7 promoter
and would that therefore be sense strand of my insert?!?

I am most likely thinking about this way too much , please help!

-mbarney-

QUOTE (mbarney @ Aug 16 2006, 08:10 AM)
Thanks for that.

I will take the promoter PCR approach, but still confused about the orientation of my insert in the vector...
If my insert is lying 3'-5' then do I design the promoter sequence and gene specific primer to read 5'-3' or as the insert lies 3'-5'.

Also if my insert is lying 3'-5' is that technically the antisense strand meaning that if I carried out IVT on my plasmid as is i would produce a strand that reads
(Insert 5'-3')-T7 promoter
and would that therefore be sense strand of my insert?!?

I am most likely thinking about this way too much , please help!


Even if your insert is orientated in the wrong direction it irrelevant once you start your PCR. You still have the 'sense' strand there so if you design two primers, one with the T7 promoter followed by the being 18-20 bp of your sense strand and a second primer with the complement to the last 18-20 bp of your antisense strand you'll get a linear fragment that looks like:

T7 promoter-5'-insert-3'

There'll be no run on because you've created a terminated linear fragment of defined length.

-Doc_Martin-