Q: Trouble with Kindergarden Ligation (SspBI/ApaI)

brett brett at BORCIM.WUSTL.EDU
Mon Sep 22 08:21:10 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!

Well, maybe you took steps to avoid Taq activity in your resriction digests of
the PCR.  If not, I'd check that out...SDS/ProtK treat (yes, this is important),
phenol extract, EtOH ppt, then digest. 
Brett Lindenbach
brett at borcim.wustl.edu

Dept Molecular Microbiology
Washington University School of Medicine
Box 8230
660 S. Euclid Ave
St Louis, MO 63110

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