cohesive and blunt ligation protocols

austin at camtwh.eric.on.ca austin at camtwh.eric.on.ca
Wed May 24 19:10:22 EST 1995


In article <3ptu0c$kpa at netnews.upenn.edu> Aron Jaffe <jaffe at a1.mscf.upenn.edu>
 writes:
>jculi at mvax.cbm.uam.es (Joaquim Culi ) wrote:

>>  Have you ever tried to ligate a fragment with one compatible cohesive
>> termini and another non compatible? I've been trying for two weeks and I
[another suggestion snipped]

try playing around with the ATP concentrations in the ligation mixture...
and this also includes looking at the concentration in the 10x stock
buffer you might be using.

if your [ATP]>0.5mM, blunt-end ligations may be inhibited, which is not
the case for 5' or 3' overhangs (these differ too BTW).

Bottom line: titrate the ATP concentrations for your particular ligation
from say 0.5 to 2.0 mM ATP and see which one works for your reaction.

Good luck,

austin so (hae-jin)
austin at camtwh.eric.on.ca




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