help: ligation xho1 problems

suter at de.d400.mpg.MPIZ-KOELN.VAX suter at de.d400.mpg.MPIZ-KOELN.VAX
Mon Sep 20 11:48:57 EST 1993



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



More information about the Methods mailing list