Building T7 promoters onto primers

Dr. Duncan Clark

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Sep 21, 1998, 3:00:00 AM9/21/98

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Hi Folks,

I need to run a transcript off a 1kb PCR product purely as a means to
generate RNA as a test system for an RT reaction.

I know I can just stick a T7 promoter sequence on the 5' end of one of
my primers but is there any additional extra sequence after the promoter
that would aid/increase the amount of RNA produced? Just wondering. I'll
probably use an Ambion or Epicentre transcription kit.

Many thanks

Duncan
--
The problem with being on the cutting edge is that you occasionally get
sliced from time to time....

Duncan Clark
DNAmp Ltd.
Tel: +44(0)1252376288
FAX: +44(0)8701640382
http://www.dnamp.com
http://www.genesys.demon.co.uk

Ashok Aiyar

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Sep 21, 1998, 3:00:00 AM9/21/98

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On Mon, 21 Sep 1998 12:51:34 +0100,
Dr. Duncan Clark (dun...@nospam.demon.co.uk) wrote:
>I know I can just stick a T7 promoter sequence on the 5' end of one of
>my primers but is there any additional extra sequence after the promoter
>that would aid/increase the amount of RNA produced? Just wondering. I'll
>probably use an Ambion or Epicentre transcription kit.

I did this a few years ago to produce RNAs corresponding to the
5' end of retroviral RNAs, and it works quite well. My recollection
is that I used just a 17 bp T7 promoter, and ensured that the +1
residue was a "G".

There is a good discussion of the template requirements for T7 RNA
polymerase in a methods in enzymology chapter by Milligan and
Uhlenbeck, which I have listed below.

Milligan, J.F., and Unlenbeck, O.C. (1989) Synthesis of small RNAs
using T7 RNA polymerase. Methods in Enzymology 180:51-62

Ashok
--
Ashok Aiyar, Ph.D.
McArdle Laboratory for Cancer Research
ai...@ebv.oncology.wisc.edu

Rich Dudley

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Sep 21, 1998, 3:00:00 AM9/21/98

to Dr. Duncan Clark

Duncan--

Sorry I don't have the complete story in front of me, but here are a few
refs I've found and some ideas:

1) Kozak sequence. This won't make more RNA, but it will make for more
efficient RNA translation. Somewhere in these archives is a couple of
good messages about Kozak sequences.

2) A post from Paul Hengen dated 27 Oct 1997 has some good info about
the sequence of T7 promoters. The consensus is the same one used by
Invitrogen, so you can look in their catalog also. Again, this makes
for more efficient RNA.

3) There is some spacing that favors increased transcription. However,
I don't have the refs in front of me. I found them in PubMed at NCBI's
website

I'll let you know if I can excavate anything else.

rich

--- --- --- -- -- -- --- --- ---
Richard J. Dudley (rdud...@pitt.edu)
Research Specialist V
Dept. of Cell Biology and Physiology
University of Pittsburgh
http://www.cbp.pitt.edu
---> search BIONET archives at http://www.bio.net <---

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ELA...@ambion.com

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Sep 23, 1998, 3:00:00 AM9/23/98

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In article <LT7aVCAG...@genesys.demon.co.uk>,

"Dr. Duncan Clark" <dun...@genesys.demon.co.uk> wrote:
> Hi Folks,
>
> I need to run a transcript off a 1kb PCR product purely as a means to
> generate RNA as a test system for an RT reaction.
>

> I know I can just stick a T7 promoter sequence on the 5' end of one of
> my primers but is there any additional extra sequence after the promoter
> that would aid/increase the amount of RNA produced? Just wondering. I'll
> probably use an Ambion or Epicentre transcription kit.
>

5'-TAATACGACTCACTATAGGG(AGGAGG) Add this to the 5' end of your 5' primer to
make a template for sense strand RNA. You can use the PCR product in the
transcription reaction without further purification.The first transcribed
base is the middle G in the triplet before the parenthesis. The bases in the
parenthesis are optional . We add them so there is no C or U transcribed in
the first couple of bases. When making a high specific activity C or U
labeled probe, this helps avoid premature termination (no Clinton jokes
please).

Eric

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Dr. Peter Gegenheimer

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Sep 25, 1998, 3:00:00 AM9/25/98

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On Wed, 23 Sep 1998 15:41:25, ELA...@AMBION.COM wrote:

> 5'-TAATACGACTCACTATAGGG(AGGAGG) Add this to the 5' end of your 5' primer to
> make a template for sense strand RNA. You can use the PCR product in the
> transcription reaction without further purification.The first transcribed
> base is the middle G in the triplet before the parenthesis. The bases in the

<snip>

The first nucleotide of the RNA transcript corresponds to the _first_ G in the
above-mentioned triplet, i.e. the G immediately following ...ACTATA.

Tan Tien Chye

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Sep 26, 1998, 3:00:00 AM9/26/98

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I am trying to do something similar. I was looking at the possiblity of
using LITMUS cloning vector from NEW ENGLAND BioLabs.

You should be able to get some info from their web-site at
http://www.neb.com
Or check out this reference "Evans, P.D. et al. (1985) Biotechniques 19,
130-135 "

Hope this stuff is useful.

Do let me know how it works out. Thanks

Tien Chye
National University of Singapore
> ----------
> From: Dr. Duncan Clark[SMTP:dun...@nospam.demon.co.uk]
> Reply To: Dr. Duncan Clark
> Posted At: Monday, September 21, 1998 7:51 PM
> Posted To: methds-reagnts
> Conversation: Building T7 promoters onto primers
> Subject: Building T7 promoters onto primers

>
> Hi Folks,
>
> I need to run a transcript off a 1kb PCR product purely as a means to
> generate RNA as a test system for an RT reaction.
>
> I know I can just stick a T7 promoter sequence on the 5' end of one of
> my primers but is there any additional extra sequence after the
> promoter
> that would aid/increase the amount of RNA produced? Just wondering.
> I'll
> probably use an Ambion or Epicentre transcription kit.
>