Linker interferes w Transformation?

Duncan Clark Duncan at genesys.demon.co.uk
Fri Jun 17 06:03:02 EST 1994


In article: <2tq04k$94t at news.bu.edu>  rocket1 at bu.edu (Richard Near) writes:
> 
> 
> 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?
> 

A few things. I assume the cells were competent ie a control was run. 
Does the linker self-ligate - use PAGE? Does the plasmid ligate and transform
OK without linker 'cos you don't mention dephosphorylating the vector? 
The amount of linker you are using seems rather excessive! 

Duncan 

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Duncan Clark                        | Internet:    duncan at genesys.demon.co.uk
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