In article <2vf5tj$291 at canopus.cc.umanitoba.ca>, sowa at cc.umanitoba.ca
(Aleksander Sowa) says:
>>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
>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.
Did you dephosphorylate the vector after blunting?
>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).
This may depend on the E.coli stab which you use to propagate the
vector (try DH5a)
>Are there any other binary plasmid available which I could use to clone
>insert in and transform tobacco??????
>>Thanks in advance for any help and suggestions!!