Ligation of large plasmids
bkodrzyc at sumter.awod.com
Fri Mar 31 11:14:09 EST 1995
Anyone have any tips on making large plasmids? We are having trouble
constructing a double insert plasmid totaling about 10 kb in size. The
first insert of 3 kb goes in without a problem. However, addition of an
additional 4 kb hunk of DNA is proving to be very difficult. I know that
efficiency of ligation goes down with increasing plasmid size but this is
a forced cloning with Hind III and Kpn I ends so logically it should work.
Also for some reason insertion of a double blunt piece is very difficult
at times. We are currently using GIBCO/BRL ligase buffer and New England
Biolabs T4 DNA ligase.
Any input would be appreciated.
bkodrzyc at awod.com
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