Q: Trouble with Kindergarden Ligation (SspBI/ApaI)
wgschech at med.uni-tuebingen.de
Mon Sep 22 04:46:26 EST 1997
Hi you all molbio gods and experts!
Now I'm spending more than two weeks with a simple PCR-based
muatgenesis using PCRing a 400bp fragment (using PowerScript from
PAN systems, it's like Pwo or Pfu). I simply can't insert a sticky
fragment: I'm using two primers (one carries the mutation) to
generate the PCR fragment. Each primer has a
restriction site at one end (SspBI and ApaI), flanked by four
additional bases (...GGGCC|C-TATT (5' overhang) for the ApaI site,
GAGC-T|GTACA... (3' overhang) for the SspBI site). The fragment I get
should be that what I wanted to amplify, I tested this with a digest.
Then I cut out the SspBI-ApaI fragment from the plasmid where I want
to introduce the mutation, the PCR fragment is trated in the same way
to get sticky ends. The fragments the are gel purified and ligated
(T4 ligase, overnight at room temp). Then: either no colonies or as
much as on the control plate (excised vector only).
What's happening here? Actually this shouldn't be a problem for
molbio beginners in their first lesson. Have you any ideas?
Could it be that four bases are not enough for the enzymes to find
and cut their sites?
All input is welcome!
University of Tuebingen
email: wgschech at med.uni-tuebingen.de
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