PCR using M13 uni & reverse primers

Michelle Gleeson michelle at MOLECULE.BIO.UTS.EDU.AU
Sun Oct 20 18:06:17 EST 1996


G'day Harry,

I use M13 primers all the time for this purpose.  The primer sequences
are:
M13 Forward (-40) 5' GTT TTC CCA GTC ACG AC 3'   AND
M13 Reverse       5' CAG GAA ACA GCT ATG AC 3'  (both 17 mers)

Make sure the suspension of bacteria/sdH2O is just barely turbid, boil for
5min, then snap chill on ice.  I make a 100microlitre suspension and use
5microlitres as a target in a 50microlitre PCR reaction, using a "standard"
program. With a smaller volume of initial suspension it is too easy to be
heavy handed with the bugs.
 In estimating the size of the insert, you need to take into
account the distance of the primers up and downstream of the insert, which
depends on the vector.

Good luck,

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Michelle Gleeson                      Thought for the day...
Molecular Parasitology Unit     Remember that the toes you step on today
University of Technology        could well be attached to the legs that
Sydney AUSTRALIA                support the arse you need to kiss tomorrow
michelle.gleeson at uts.edu.au
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On Sun, 20 Oct 1996, Harry Witchel wrote:

> Felicitations Molecular Mages --
>    I am interested in using the M13 uni and reverse primers to amplify
> inserts in vectors which have both Uni and reverse -- I would use entire
> bacteria as template (boiling the bugs first).  I have tried this with
> one set of uni and reverse primers, and it did not work.  A heard that
> the success of this amplification depends explicitly on precisely which
> Uni and reverse primers one chooses.  Does anyone have any succesful
> experience with this technique?  I was going to use:
>
> M13 forward -- 24 bases
> CGC CAG GGT TTT CCC AGT CAC GAC
> Tm=3D 73.6=B0C
>
> m13 rEVERSE -- 23 bases
> AGC GGA TAA CAA TTT CAC ACA GG
> Tm =3D 62.3=B0C
>
> Many thanks,
> Harry
>
> Harry.Witchel at Bristol.Ac.Uk
>
>
>




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