help: ligation xho1 problems

Michael G. Kurilla mgk2r at Virginia.EDU
Mon Sep 20 10:35:46 EST 1993


suter at de.d400.mpg.MPIZ-KOELN.VAX writes:
> 
> 
> for several weeks now, I have been trying to clone a lambda fragment over XhoI.
> I have now simplified the procedure, still I am unable to ligate. do any of
> you have useful ideas, or run into the same kind of problem ?
> 
> what i do is:
> 1. digest 1 microgram pBluescript with 5 units XhoI (several suppliers tested),
>  all digests in Boehringer buffer 'H'
> 2. after 1 h at 37C, heat 20 minutes at 65C, cool to RT, enlarge volume 5-fold
> 3. add ligase in buffer (several suppliers), incubate at RT, 2 hours
> 4. test on gel to observe ligation, compare to dummy reaction without ligase
> 
> result: wherease i can succesfully digest my DNA with both XhoI and several
> other enzymes (EcoRI, BamHI, SalI, HaeIII, HindIII, Asp718, Apa1), i am 
> absolutely unable to re-ligate the XhoI reaction ! Help !
> 
> distressed, clemens
> ===============================================================================
> Dr. Clemens Suter-Crazzolara, PhD
> Max-Planck-Institut fuer Zuechtungsforschung
> Abteilung Genetische Grundlagen der Zuechtungsforschung
> Carl-von-Linne Weg 10,        D-50829 Koeln, Germany
> Tel.xx49.221.5062-221    Fax.-213      e-mail: suter at vax.mpiz-koeln.mpg.d400.de
> ===============================================================================
> 	if this is victory, our hands are to small to hold it

I have seen this problem with XhoI in the past.  A call to NEB
offered some good advice.  They claim that XhoI can become
covalently attached to the cut ends of the DNA.  Phenol
extraction can then result in some loss of DNA.  They
recommended digesting with proteinase K after incubation, then
phenol extraction.  Their advice did result in successful
cloning.

Mike Kurilla



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