plasmid construction using adapters.
bsh at MED.PITT.EDU
Sat Jul 9 12:25:45 EST 1994
ckafer at harp.aix.calpoly.edu (Christopher W Kafer) writes:
>We would like to insert a DNA fragment into a vector in which the two have no
>restriction sites in commoni.e.1 has a BamHI overhang the other a NcoI overhang
>What I would like is a protocol or advice on joining 2 noncompatible cohesive
>ends using adaptors. The only protocols I have found are for using adaptors to
>add restriction sites to blunt end fragments.
>We have the adaptor,two unphosphorylated ssDNA oligos in separate vials. Where
>would you suggest I go from here? Any advice will be greatly appreciated as we
>are somewhat inexperienced at constructing our own plasmids. Responding by
>either email or posting is fine. Thanks in advance.
I would first kinase the adapters, mix them in equimolar amounts in 25 mM
Tris, pH 8, 10 mM MgCl2, to a final concentration of 20 uM. Heat to
95C for 5 min, allow to cool to rt over about 30 minutes by doing this
incubation in a heating block and letting it come to room temp. Ligate,
digest with the enzyme that would make sure you have only one adapter
molecule per insert, do a standard PEG precipitation to remove the unligated
adapters, and now ligate it to the plasmid molecule. You should be able
to accomplish this also by ligating the adapter first to the plasmid and then
ligating with the insert. If you need more details, I can guide you to some
bsh at med.pitt.edu
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