Insert size?

Martin Kennedy mkennedy at chmeds.ac.nz
Tue Nov 23 18:57:16 EST 1993


In article <2ct5k7$dct at gazette.bcm.tmc.edu>, jamess at mbcr.bcm.tmc.edu (James S. Sutcliffe) writes:
> 
> Yet another followup to this insert size issue:
> 
> Tony was quite correct in his statement about the correct molar ratios of
> the fragment ends. Another thing that might help would be to do a two step
> ligation, where the first "step" is a short (e.g. 2-3 h) at a higher
> concentration to promote intermolecular ligation events and the second
> "step" is at a more dilute concentration to promote intramolecular ligation
> events. Don't forget that transformation efficiency is inversely
> proportional to the size of the construct. Hope this helps.
> 
> Jim Sutcliffe

On this note, you can transform E.coli with lambda DNA, but the efficiency is
lousy - 10^3 or 10^4 per ug of DNA.  I used to work on F plasmid, and I never
remember successfully transforming it into a cell (it is ca 100kb).  So even a
native, large plasmid is likely to be tough to get into E. coli.  Combine 
this difficulty with the inefficencies of generating effective recombinants 
inherent in any ligation and I think you have the answer to the problem of
cloning big fragments in plasmids.  Don't forget too that packaged lambda
recombinants infect the cell, they aren't transfected into it, so they aren't 
             ^^^^^^
subjected to the constraints of plasmid transformation systems.
 

-- 
Cheers,

Martin

NNNN   NN  Martin A Kennedy (E-mail = mkennedy at chmeds.ac.nz)  ZZZZZZZ  
NN NN  NN       Cytogenetic and Molecular Oncology Unit          ZZZ
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