Repost of request in plain text.

Bernd Weisshaar weisshaa at mpiz-koeln.mpg.de
Fri Oct 15 04:09:08 EST 1999


Hi,

we had good results with lambda DNA from Qiagen preps. However, also 
that was not really stable. To me, it seems that this DNA is not 
sufficiently pure. I recommend to our users to do PCR on the lambda 
DNA. We use the resulting PCR fragment as template, and that worked 
quite well for us. The drawback are problems with large genomic 
inserts, but even in such cases two outward primers from the probe 
combined with vector-flanking primers help sometimes.

We use the PE "1000bp protocol" provided by the PE people at 
Weiterstadt, and that works really well. But the instrument is 
occupied a long time, and you have to play tricks to increase data 
collection time.

Regards, Bernd



>Dear all,
>
>I am working with 377 for lambda DNA sequencing.  I have lots of question
>about my sequencing work, for example:
>
>1. How I can make 48 cm running to read 700 to 900 base, (now I can read
>only 400-500 base)?
>2. I am using the Wizard Lambda DNA purification system from Promega to
>prepare my template for Big-Dye Terminator reaction and the sequencing
>results are not very stable, sometime is reasonable, and sometime is very
>bad. Does anybody know how to deal with this sequencing work?
>
>Thank you for your help in advance.
>
>Rui Mang
>Department of Human Retrovirology
>AMC, University of Amsterdam
>Meibergdreef 15
>1105 AZ, Amsterdam
>The Netherlands
>Tel: 31-20-5664522
>Fax: 31-20-6916531
>Email: r.mang at amc.uva.nl

_____________________________________________________________________

PD Dr. Bernd Weisshaar
MPI fuer Zuechtungsforschung
Carl-von-Linné-Weg 10
50829 Koeln
Germany

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