IUBio

Sequening bacs with big dye chemistry?

Bernd Weisshaar weisshaa at mpiz-koeln.mpg.de
Mon May 4 04:31:09 EST 1998


>I am looking for information on sequencing bac's with regard to the new
>'big dye' chemistry of the ABI Prism 377.  How do you prepare your
>template, cycle sequence reaction, etc.  I have been unsuccessful in my
>attempts with stardard ABI protocols with regard to template and primer
>concentrations.

Dear Mairead,

we have used the following:

about 12 microl volume,
8 microl premix
10 pmol primer
3 microg DNA
- 4' 98 C
- 30 cycles

In principal, it worked. Our users have used Qiagen colums for template
purification. Still, some people get better DNA than others. As usual (or
here even more), template quality is crucial.

Best regards, Bernd

PS: In case that you get more personal replies, could you post a summary =
to
the net? BW

_____________________________________________________________________
PD Dr. Bernd Weisshaar
MPI f=FCr Z=FCchtungsforschung
Carl-von-Linn=E9-Weg 10
50829 K=F6ln
Germany

Tel. office: +49-221-5062 590   Tel. lab: +49-221-5062 311
Fax: +49-221-5062 313
e-mail: weisshaa at mpiz-koeln.mpg.de
WWW-URL: http://www.mpiz-koeln.mpg.de/~weisshaa/BW-info.html





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