Validating 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.
Genomic
DNA extraction from Arabidopsis
leaves
Sow
about twenty seeds on soil, and grow plants until the rosettes have formed.
- Grind one leaf in liquid
nitrogen
- Add 400 µl
extraction buffer and mix well
- Incubate 15 min at
50°C
- Centrifuge 15 min at
4°C, 4100 rpm
- Transfer 150 µl to
a new tube
- Add 150 µl of
isopropanol
- Vortex and incubate 20 min
at room temperature
- Centrifuge 30 min at
4°C, 4100 rpm and pour off the supernatant
- Add
125 µl
of isopropanol and centrifuge 15 min 4°C at 4100 rpm
- Pour off the supernatant and
dry the pellet
- Resuspend the pellet with
125 µl of TE (10mM, 1mM) pH 7.5
- Use 2 µl for PCR
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
| 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 |
