Cloning into PBI121 binary plasmid

Andreas Weber aweber at biolan.uni-koeln.de
Thu Jul 7 11:26:04 EST 1994


In article <2vf5tj$291 at canopus.cc.umanitoba.ca>, sowa at cc.umanitoba.ca 
(Aleksander Sowa) says:
>
>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.

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 
my
>insert in and transform tobacco??????
>
>Thanks in advance for any help and suggestions!!
>
>Alek
>




More information about the Methods mailing list