SmaI site gone!?
espinoza at cgl.ucsf.edu
Mon Sep 22 12:57:50 EST 1997
BMBTJY at leeds.ac.uk (T.J. Young) writes:
>A strange thing has happened. I subcloned a cDNA sequence into a eukaryote
>expression vector using flanking SmaI restriction sites. I've used this
>construct for expression and everything seems to be working fine. However,
>I've done some SDM on the gene in this vector and I now want to subclone it
>into a different plasmid. Well, seeing as I ligated it in at SmaI sites I
>thought I would excise it at the SmaI sites but they appear to have gone!?
>Now the only thing I can think of is that I had some exonuclease activty
>arround when I did the ligation which has chewed back the SmaI recognition
>site. Is this something other people have seen? Or could there be another
>explanation? Thanks in advance.
SmaI does that. I don't know why, exactly (contaminating exonuclease?
SmaI is an exonuclease?), but SmaI will drop base pairs. It has happened
to me twice (one unexpectedly led to a neat new allele 8-) ), so I never
use SmaI to clone with if I can avoid it (XmaI will substitute effectively
most of the time)
-Hernan, SmaI is the devil's enzyme, never turn your back on it. 8-)
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