Subcloning Poly-Linkers

J Preiss--Seq Anal preissj at CLVAX1.CL.MSU.EDU
Tue Dec 8 14:48:00 EST 1992


Howdy Net-Folk

	Got a small cloning problem.  I'm trying to construct a plant
promoter testing T-DNA vector.  I have a binary T-DNA with a GUS reporter
gene/NOS terminator with no promoter and a single HindIII site to drop
in a putative test promoter.  I constructed it for my thesis work.  Now
I want to test some other putative promoter fragments that have internal 
HindIII sites, so I deleted some miscelenious restriction sites around the
T-DNA vector and want to add the pBLUESCRIPT MCS.  I have deleted the HindIII
site from pBLUESCRIPT-II-SK+ and saved this new plasmid.  Next I cut out
the MCS with BssHII, flushed with T4-Pol, ligated on HindIII linkers, cut
with HindIII, and tried to ligate into the HindIII site in the T-DNA.  I
don't get any possative clones. 

Questions:
1.	Does anyone know any peculiar problems with cloning poly-linkers?

2.	Does anyone know any tricks to doing the above?

If I get some functional suggestions I will summarize (and try to make 
the T-DNA vector available).  If no suggestions, I will just try again.
Thanks in advance.	
		
		Lenny Bloksberg
		PreissJ at clvax1.cl.msu.edu




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