Problem with lambda subcloning and sequencing

Xavier Gansel Xavier.Gansel at unifr.ch
Fri Oct 11 10:28:02 EST 1996


Hi,

The aim of this question concern sequencing of lambda DNA.

I cloned a genomic fragment in a lambda phage corresponding to the cDNA I
am studying. The genomic part corresponding to the ORF have been subcloned
and sequenced and I try to go in the promoter walking form the ORF.
It turns out that I can't subclone any fragment of this promoter in pUC
based plasmid by walking from the 5' end of the ORF. I mean that all the
transformant I got are shorter than expected, when I obtain some, and do
not hybridize with my probe.
I tried to cycle sequence "by hands" from lambda DNA with no results, and I
do not have access to an automated sequencer.

I want the sequence of this promoter in order to analysis is regulation ...
any advices would be greatly appreciated,
Thanks in advance,

Xavier.

Could you please directly reply to my e.mail address, I shall post back a
summary of your answers.


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Xavier Gansel
Plant Biology Institute
University of Fribourg
3 Rue A. Gockel			tel      +41 37 29 88 18 / 10
1700 Fribourg			fax      +41 37 29 97 40
Switzerland			e-mail   xavier.gansel at unifr.ch

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