Q: Trouble with Kindergarden Ligation (SspBI/ApaI)

[Wolfgang Schechinger]

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!

Wolfgang
-----                              
Wolfgang Schechinger         
University of Tuebingen
email: wgschech at med.uni-tuebingen.de
http://www.medizin.uni-tuebingen.de/~wgschech/research.htm
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