Sequencing Artifact in two-hybrid bait plasmid

Angela Manning a.manning at ualberta.ca
Thu Feb 27 17:22:34 EST 1997


Dear Fellow two-hybrid screeners,

We have made a primer that allows sequencing through the HA tag, cloning
site and insert of the two-hybrid bait plasmid pAS1-CYH2.  From the
published sequence, there is a run of C's and G's near the BamHI site as
follows:

     5'-...GGCCCCGGGGATCC...-3' (two G's, four C's, four G's).
             ^^^^

With both Sequenase 2.0 and Thermo-Sequenase, we get an electrophoresis
artifact that causes the sequence to look like only three C's are present
after the two G's.  Repeated sequencing with deoxy-Inosine shows that there
are really four C's there, so there is no real cause for alarm (i.e.
thinking your bait constructs are out-of-frame).  We don't know how an
automated sequencer would call the sequence but suspect it may only see
three C's.

Has anyone else seen this, or found any other problems with these plasmids?
 
Regards,
Morris

-- 
Morris Maduro       maduro at odum.biology.ualberta.ca
Dept of Biological Sciences
University of Alberta
Edmonton, AB Canada
T6G 2H3



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