DNA sequence puzzle!

aEscher at ccmail.llu.edu aEscher at ccmail.llu.edu
Mon Mar 20 13:07:48 EST 1995


A student came to see me with a strange result from a DNA sequencing
gel. He sequenced a gene cloned into bluescript SK- (sequencing was done
 on ds DNA using sequencing grade Taq polymerase from Promega).The gene
was cloned as a XbaI-XhoI fragment, with the AUG start codon downstream
of the XbaI site. He used a T7 oligo to sequence the 3' end of the cloned
gene, but what he got instead was the 5' end of the non coding strand in 
a reverse orientation! i.e., the published coding strand is 5'...TCTAGA..
..ATG..3' and instead he obtained 5'..AGATCT...TAC....3'! This phenomenon 
has happened with another gene in another vector as well. Are we missing
something obvious that is going to make me feel really stupid? No matter
what kind of mistake was made in cloning, choice of oligo etc, we just
should not get the noncoding strand changed from a 3' to 5' direction
into a 5' to 3' orientation. Suggestions?
Thanks
Alan Escher.

but he got 5'...



More information about the Methods mailing list