Using TdT to clean up sequencing reactions?

drdad at ktc.com drdad at ktc.com
Tue Aug 27 11:41:17 EST 1996


In article <drm21-2108961532030001 at nntp-serv.cam.ac.uk>,
drm21 at mole.bio.cam.ac.uk (David Micklem) wrote:

> In article <drdad-2008961720070001 at 10.3.2.45>, drdad at ktc.com wrote:
> 
> 
> >Hello,
> >
> >I've tried the TdT technique and really prefer the Klenow co-sequencing
> >method referred to in Biotechniques Vol 17, pg. 286 (1994). I use this as
> >part of my routine sequencing protocol now.
> >
> >Good luck!
> >
> >Felix Guerrero
> >drdad at ktc.com
> 
> Hi Felix,
> 
> I'm interested in what this protocol is.  Could you post a summary? (We
> can only get the totally useless Euro-version of Biotechniques here). I've
> toyed with trying the TdT method, but have never actually gotten round to
> buying any.  Klenow, on the other hand, we have lots of! 'Co-sequencing'
> also seems to imply that there are no additional steps involved. Is this
> correct?
> 
> David


Hello,

The Klenow co-sequencing is surprisingly very simple. I use the standard
Sequenase Ver. 2.0 protocol with the only change being that after adding
the Sequenase enzyme to the DNA-annealled primer/buffer solution, one adds
1 unit of Klenow. Then the only difference is to allow the termination
reaction step to proceed at 37 degrees C for 45 minutes instead of the
usual 2-4 minutes.

Pretty easy and I think there is a noticeable improvement in band
uniformity from difficult templates. At times, I have had to resort to
dITP sequencing, but now I add the Klenow routinely!

Good luck,

Felix



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