Cloning into PBI121 binary plasmid

Aleksander Sowa sowa at cc.umanitoba.ca
Wed Jul 6 15:58:27 EST 1994


Hi netters!

I have been trying to clone blunt-ended insert into Xba1 and Xba1/Sst1
sites of PBI121 binary plasmid (Clontech). Xba1 allows to clone between
CaMV35S promoter and GUS whereas digest with Xba1 and Sst1 cuts GUS out of
the cassete leaving CaMV35S promoter and NOS terminator. 
I have been using Klenow, T4 DNA Polymerase and Mung Bean Nuclease to
blunt-end vector and then T4 DNA Ligase to ligate with insert.
So far no results.......
Did anyone have similar problems??? Any suggstions how to solve it?

I have also found that Sma1 and EcoR1 sites were not unique in this
plasmid (as opposed to Clontech's wiew). 
Are there any other binary plasmid available which I could use to clone my
insert in and transform tobacco??????

Thanks in advance for any help and suggestions!!

Alek




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