In article <2vf5tj$291 at canopus.cc.umanitoba.ca> sowa at cc.umanitoba.ca
(Aleksander Sowa) writes:
>>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??????
>I've also used pBI121 vector for my transformation experiment. Overall feeling
is that it's not easy, but is doable:
(1) As suggested by another netter, dephosphorylate the vector after blunting.
I used BamHI/SacI, then Klenow/T4 DNA Pol to blunt, followed by CIP to de-
phosphorylate. This reduced the number of colonies I have to screen before
obtaining the correct construct.
(2) Still need patience. Once I did 24 mini-prep, only one turned out to have
insert. To obtain sense and antisense constructs, I screened 72 colonies before
luck struck me.
Hope this may help in any way