Concentration of DNA in Ligations???

David R. Johnson johnson at biomed.med.yale.edu
Tue Jul 30 13:47:14 EST 1996


> M C Behrendt (M.C.Behrendt at shef.ac.uk) wrote:
> : Does anyone know what the optimum concentration of DNA in a ligation
> : reaction is (NOT the ratio of vector to insert), and what the ideal volume
> : per eppendorf should be.  At the moment I'm using approx 3-4ug of DNA (insert,
> : 660bp plus vector, 3674bp, 2:1 ratio) and 1-2 units of Ligase (+ buffer, from
> : Gibco) in a total volume of 100ul which is aliquoted out in 10ul amounts for
> : an overnight incubation at 11 or 16C.  My problem is that I need this to work
> : with a very high efficiency in order to construct a library - but - it's not
> : working.
Chia Jin Ngee wrote:
> Optimum total DNA concentration should be 1 ug/ml. Ideal volume ranges
> from 10 to 30 ul. Usually 1 unit of ligase is enough for all ligations.
> Blunt ended ligations may require 5 to 10 units.

dear ligators (here's my 2nd hand $0.02)
   A nice (and free!) analysis of this was published in the GIBCO/BRL 
publication "Focus" at some point in the distant past (ok, probably 5+ 
years ago).  
they said:
   20 fmol vector + 60 fmol insert in 20 ul ligation.  Naturally, they 
recommended using what is (probably) an excess of ligase and a buffer 
that included PEG (to help concentrate DNA, buffer is now included with 
the ligase).
   I use half this volume and transform with 2ul, leaving a lot for a 
gel in the unlikely event that it is necessary (to help figure out why 
if the transformations aren't what they should be).  

Talking in terms of mass (ug) is potentially misleading since vectors 
vary so much in size.  Similarly, concentration of DNA ends is what is 
critical, not total DNA concentration. 
  Here's to getting it together! 
  see you later, eh ligators?
  -Dave



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