The AGRIKOLA consortium has cloned most of the CATMA GSTs into pAGRIKOLA, a Gateway® destination vector based closely on the Hellsgate 12 vector developed by Chris Helliwell, Peter Waterhouse and colleagues. These expression clones are in E. coli DH5α. pAGRIKOLA confers resistance to kanamycin in bacteria.
The Gateway® cloning within the AGRIKOLA project employed high-throughput protocols that avoided isolation of single colonies during the cloning procedure. Therefore, before going any further you should streak the bacteria out on agar plates containing 50 mg.l-1 kanamycin and select 4 or 6 individual colonies for verification.
Knowing the size of your chosen GST, you can verify the plasmid by restriction digest using the pAGRIKOLA restriction map or better still by PCR amplification and/or sequencing. We use the primers shown below for validating pAGRIKOLA clones.
Agri 51 : 5' CAA CCA CGT CTT CAA AGC AA 3'
Agri 56 : 5' CTG GGG TAC CGA ATT CCT C 3'
Agri 64 : 5' CTT GCG CTG CAG TTA TCA TC 3'
Agri 69 : 5' AGG CGT CTC GCA TAT CTC AT 3'During the Gateway® recombination reaction, approximately half of the clones end up with an inverted intron cassette, i.e. the 1st GST will amplify with primers Agri51 and Agri64 and the second with primers Agri56 and Agri69. This has no noticeable effect on the efficiency of silencing when these constructs are introduced into plants (the CAT intron will be spliced out instead of the PDK intron).
|mix 1||mix 2||mix 3||mix 4|
|Agri51 10 µM 0.2 µl||Agri51 10 µM 0.2 µl||Agri56 10 µM 0.2 µl||Agri64 10 µM 0.2 µl|
|Agri56 10 µM 0.2 µl||Agri64 10 µM 0.2 µl||Agri69 10 µM 0.2 µl||Agri69 10 µM 0.2 µl|
|10 x Red Cresol (Aldrich cat no. 11447-2)||2 µl|
|10x PCR buffer||2 µl|
|dNTP stock (2,5 mM of each dNTP)||0.8 µl|
|Taq enzyme (home made...)||0.2 µl|
|35 X||55°C||30 sec|
mix 1 (agri51/agri56) = 245 pb + gst length
mix 2 (agri51/agri64) = 442 pb + gst length
mix 3 (agri56/agri69) = 145 pb + gst length
mix 4 (agri64/agri69) = 342 pb + gst length