Question on cloning

"udo wegmann IFR udo.wegmann at BBSRC.AC.UK
Thu Jan 18 04:37:38 EST 2001


Hi,
you could digest 50% of your vector with EnzymeI and 50% with EnzymeII and
run a gel afterwards to check that they have both worked. Than go ahead with
the second digest. Assuming that at least one of your enzyme works well
close to the end of a dna molecule 50% of your vector will be cut properly.
Your results so far could indicate that one of your enzymes does not work
very well close to the ends of a dna molecul. You can check that out in the
NEB-catalogue appendix. They have a section were they have checked that for
some vectors and short oligonucleotides (p.210,211). Furthermore I would
suggest that you think about a recut after ligation to get rid of the
religated vector. You might find a suitable restriction site (which is not
present in your ligation product) in the vector region between the two
restriction sites you are using to clone your insert. (Sorry, if you have
thought about that already).

Good luck Udo



DR. Udo Wegmann
Department of Food Safety Science
Institute of Food Research
Norwich Research Park
Colney, Norwich NR4 7UA
Tel.: +44/(0)1603/255252
E-mail: udo.wegmann at bbsrc.ac.uk


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