J Preiss--Seq Anal
preissj at CLVAX1.CL.MSU.EDU
Tue Dec 8 14:48:00 EST 1992
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.
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.
PreissJ at clvax1.cl.msu.edu
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