Cohesive End Ligations - Question

[Ann Williams]

Kathy,
   Usually the formation of concatamers (the large polymers that will 
not enter the gel) is dependent on the concentrations of DNA ends in the 
ligation rather than any other factor.  For best results of plasmid 
(rather than lambda) cloning you want about equimolar amounts of vector 
and insert.  For a DNA fragment twice as large, you need a 4-fold 
reduction in mg/ml to keep about the same number mmoles/ml.  Even with 
the right proportions, you will encourage concatamer formation with a 
high total DNA concentration in your ligation.  Typical ligations use 
about 200ng up to 1000ng total DNA.  Keep in mind that it is not the 
concentration of DNA that is actually important, but the concetration of 
DNA ends.
  
			
Ann L. Williams
Oklahoma State University
Biochemistry & Molecular Biology
246 Noble Research Center-D
Stillwater OK 74078-3035
PH: (405) 744-9327
FAX:(405) 744-7799
email: awilliams at biochem.okstate.edu