Cutting the ends of dna

Aidan Weatherill aweather at worf.molbiolox.ac.uk
Fri Oct 29 06:02:17 EST 1999


I am building a construct by pcr'ing up 3 fragments, that I want to join
together. the pcr oligos contain restriction sites at the ends so I can cut
the products and ligate them in a sticky end way (and guarantee
orientation).

I am recovering the correct ligations by pcr'ing up from the ligation with
the most external oligos.

I have had no luck in recovering the correct construct. I believe that they
are not sticking because the enzymes are not cutting at the ends. the
enzymes are sac1 bamh1.

If someone can suggest ways of increasing the efficiency of a) cutting at
the ends of molecules and b) doing triple ligations, I would be very (very)
happy

cheers

Aidan

aweather at molbiol.ox.ac.uk





More information about the Methods mailing list