HELP WANTED: Trouble with ligation

Dr. Dr. habil U. Wernery microbio at emirates.net.ae
Thu Sep 18 11:24:06 EST 1997


I am trying to set up a genomic library of camel DNA using SK+ vector. 
Unfortunately, I have had no luck in obtaining recombinant plasmids. 

My approach involves restriction of SK+ with BamHI followed by ligation 
to 1-2 kb fragments of genomic camel DNA generated by complete digestion 
with MboI. 

Is this a suitable approach? I had set up five ligation reactions as 
follows:

Tube #1:  dephosphorylated vector only  (negative control)

Tube #2:  vector that was linearized but not dephosphorylated (positive 
control)

Tube #3:  dephosphorylated vector + 1-2 kb fragments + 15% PEG 
(polyethylene glycol)

Tube #4:  dephosphorylated vector + 1-2 kb fragment

Tube #5:  dephosphorylated vector + 1-2 kb fragments + 25% PEG

Tube #6:  dephosphorylated vector + pUC 19 control plasmid (for 
transformation efficiency determination)

The tubes were made up to volume (15 microlitres) with water, ligase 
buffer, and  T4 DNA ligase. The mixtures were incubated for 5 hours at 20 
degrees Celsius and subsequently transformed into competent DH5 alpha 
cells (from Gibco, BRL). They were then plated on LB-Amp plates and 
incubated at 37 degrees Celsius  overnight.

Examination of the plates the next day revealed that transformation 
efficiency was good (tube 6).  As expected the tube with the 
dephosphorylated vector produced no colonies (tube 1). Tube 2 containing 
the linearized but not dephosphorylated vector control produced colonies. 
This would indicate that the dephosphorylation, ligation and 
transformation controls worked. 

How can I account for the fact that no colonies were observed for 
ligation of vector  with the genomic DNA? I would be grateful if someone 
can recommend an alternative approach or suggest improvement in the above 
set up. 

Thank you very much for your time and consideration. Please reply  by 
e-mail to (Attn: Maxy Mariasegaram):

microbio at emirates.net.ae

Sincerely,
Maxy Mariasegaram





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