Linker interferes w Transformation?

Yasha Hartberg 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?
> 
> thanx
> rick
> 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.

Good Luck! 


Yasha Hartberg



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