cloning blunt

Michael D. Baron michael.baron at bbsrc.ac.uk
Fri Nov 17 05:16:52 EST 1995


In article <489lds$m9s at highway.LeidenUniv.nl>, 
nandy at ruly46.medfac.leidenuniv.nl says:
>For two months now I have been trying to clone a 2 kb blunt fragment 
(NruI/HindIII, filled in with klenow) into a PvuII site of a 6.5 kb 
vector.
>I am aware of the fact that blunt ligation are not as efficient as 
sticky end ligations, but still it should work.
>There are some specific questions I would like to put forward:
>1. Self ligation of the PvuII cut vector does not work. Is anyone aware 
of problems specific for a blunt PvuII site?

If this is so, you must suspect your PvuII or the phosphatase (I assume 
that you have used ciap?), or the ligase. I have had no problems with 
PvuII cloning (PvuI, yes, not PvuII). If the vector won't religate, one 
or more enzymes are destroying the ends of your vector, or the ligase 
isn't working. Suggest you check the ligase, and/or try different batches 
of PvuII/ciap, if you haven't already. You should get 1000's of colonies 
from 20ng vector on religation.

>2. From the catalog a learned that blunt NruI sites are difficult to 
ligate. Does anybody know why, and if there is a way to imporve this?
>
Have you considered the isoschizomer Bsp68I? Maybe NruI is simply one of 
those not very reliable enzymes, hard to get very pure, or something. 
SmaI used to have a bad reputation like this (Waaay back when).

michael baron



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