ligation problems

GIETZ at bldghsc.lan1.umanitoba.ca GIETZ at bldghsc.lan1.umanitoba.ca
Mon Sep 20 13:20:00 EST 1993


--*******--- clemens writes ---8888888888888888---------


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 !
***************************************************************************

Hi Clemens!
    I have also had problems with  ligation of an XhoI DNA fragment. It did 
go eventually, but I did a phenol:chloroform extraction and ethanol pptt
 after the Xho digestion of both vector and insert DNA.
I then purified the DNA fragment from a gel and ligated.  The frequency was
a bit lower but it did work.  Perhaps the inactive XhoI can still bind the end
of the cut DNA and affect the ligases ability to access the end.  (my best
guess)  What I would do is to phenol:Chloroform your digest,
 then ethanol pptt and then ligate after dissolving. 
I'm sure this will work, it did for me!
Good Luck!

Dan Gietz
______________________________________
R.Daniel Gietz Ph.D.
Assistant Professor
Department of Human Genetics
University of Manitoba
770 Bannatyne Ave, Rm 250
Winnipeg, Manitoba, Canada
R3E 0W3
Tel.: (204)789-3458
Fax.: (204)786-8712
E-mail GIETZ at BLDGHSC.LAN1.UMANITOBA.CA
"Trying to do the Manitoba Thing"
_______________________________________



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