Linker interferes w Transformation?
YMH4375 at zeus.tamu.edu
Sun Jun 19 15:41:04 EST 1994
In article <2tq04k$94t at news.bu.edu>, rocket1 at bu.edu (Richard Near) wrote:
> I am simply trying to insert a linker (about 10 nt, phosphorylated)
> into a plasmid in order to add a new restriction site. I have
> linearized the plasmid, filled in ends with klenow and then
> ligated (about 3 ug linker) the linker into the plamid overnite
> at 12 C. Upon transformation of DH5a (Ca competent) and selection
> on LB amp plates I got no colonies. NOW THE QUESTION: SINCE I
> DID NOT REMOVE EXCESS LINKERS AFTER LIGATION, COULD THEY
> INTERFERE WITH THE TRANSFORMATION?
> rocket1 at bu.edu
The first thing I would check is my ligation buffer. Is your buffer
optimized for blunt-end ligations? If not, USB gives a recipe that has
worked well for me in Editorial Comments, Spring '92, Vol. 19, No. 1, p.6.
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