agrikola logoValidating AGRIKOLA RNAi lines

The AGRIKOLA consortium is carrying out 4000 Arabidopsis thaliana Col0 transformations with individual pAGRIKOLA constructs. These plants have been transformed by floral dip with the strain GV3101:pMP90:pSOUP. Up to 12 transformants for each construction were selected using Basta.

Seeds from these lines are a mix of wild-type Col0, heterozygotes and homozygotes for the RNAi construct. To select plants that contain the RNAi construct, we recommend that you amplify the GSTs by PCR on genomic DNA with primers for pAGRIKOLA. Sequencing of the PCR products to validate the GST sequences is strongly recommended too.

arabidopsis plant

Genomic DNA extraction from Arabidopsis leaves

Sow about twenty seeds on soil, and grow plants until the rosettes have formed.

Extraction buffer (40 ml)

Tris HCl pH 7.5 1 M 8 ml
NaCl 5 M 2 ml
EDTA 0.5 M 2 ml
SDS 20 % 1 ml
H2O 27 ml

PCR on Genomic DNA using primers agri51/56/64/69

Agriprimers1

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'

                 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) 2.2 µl
10x PCR buffer 2.2 µl
dNTP stock (2.5 mM of each dNTP) 0.8 µl
Taq enzyme 0.2 µl
water 14.2 µl
Genomic DNA 2 µl

PCR conditions:

94°C 5 min
 
94°C 45 sec
35 X 55°C 45 sec
72°C 2 min
       
72°C 5 min

ADNg

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