4. For cloning the PCR-amplified fragment in the pDB digested
vector, follow the directions in the cloning kit manual (see
Note 10).
5. Transform competent E.coli DH5α cells with 10 μL of the
cloned PCR product in the pDB vector.
6. Plate the transformations on LB plates with Kanamycin selection overnight at 37
C.
7. Next day check colonies for gene insertion.
8. Grow two positive colonies in liquid LB with Kanamycin overnight at 37
C.
9. Next day, purify plasmids with gene of interest in pDB.
10. Sequence the gene in the plasmid to confirm no mutations and
the gene is in frame with the HA epitope tag.
11. Transform sequenced plasmid into competent Agrobacterium
GV3101 cells.
12. Plate the transformation on LB plates with Kanamycin and
Rifampicin selection at 28
C.
13. Select positive colonies for plant transformations.
3.2 Plant
Transformation
1. Grow Agrobacterium GV3101 carrying gene of interest in
pDB vector in 5 mL of liquid LB with Kan and Rif overnight
at 28
C.
2. Transfer 0.5 mL of the culture into 500 mL LB with Kan and
Rif. Grow for 16 h at 28
C.
3. Transfer 225 mL into a culture bottle and centrifuge at
5000 rpm for 10 min at 4
C.
4. Remove supernatant and repeat step 3 with 225 mL culture in
the same bottle.
5. Remove supernatant.
6. Resuspend the pellet thoroughly using pipetting and vortex in
40 mL of Arabidopsis infiltration media (see Subheading 2.2,
step 3). Once the pellet is resuspended, top up to 200 mL with
the Arabidopsis infiltration media.
7. Add Silwet L-77 to a concentration of 0.04% into the Arabidopsis infiltration culture just before ready for dipping. Mix
culture thoroughly.
8. Transfer Arabidopsis infiltration culture to a 300 mL beaker.
9. Prepare a stretch of plastic wrap ~20 cm  20 cm and spray it
with water.
10. Submerge Arabidopsis inflorescence completely for 30 s into
the infiltration media without exposing the leaves to the media.
Shake the pots while dipping into the media to make sure all
the flower buds are thoroughly exposed.
Biotin-Based Proximity Labeling of Protein Complexes in Planta
433
vector, follow the directions in the cloning kit manual (see
Note 10).
5. Transform competent E.coli DH5α cells with 10 μL of the
cloned PCR product in the pDB vector.
6. Plate the transformations on LB plates with Kanamycin selection overnight at 37
C.
7. Next day check colonies for gene insertion.
8. Grow two positive colonies in liquid LB with Kanamycin overnight at 37
C.
9. Next day, purify plasmids with gene of interest in pDB.
10. Sequence the gene in the plasmid to confirm no mutations and
the gene is in frame with the HA epitope tag.
11. Transform sequenced plasmid into competent Agrobacterium
GV3101 cells.
12. Plate the transformation on LB plates with Kanamycin and
Rifampicin selection at 28
C.
13. Select positive colonies for plant transformations.
3.2 Plant
Transformation
1. Grow Agrobacterium GV3101 carrying gene of interest in
pDB vector in 5 mL of liquid LB with Kan and Rif overnight
at 28
C.
2. Transfer 0.5 mL of the culture into 500 mL LB with Kan and
Rif. Grow for 16 h at 28
C.
3. Transfer 225 mL into a culture bottle and centrifuge at
5000 rpm for 10 min at 4
C.
4. Remove supernatant and repeat step 3 with 225 mL culture in
the same bottle.
5. Remove supernatant.
6. Resuspend the pellet thoroughly using pipetting and vortex in
40 mL of Arabidopsis infiltration media (see Subheading 2.2,
step 3). Once the pellet is resuspended, top up to 200 mL with
the Arabidopsis infiltration media.
7. Add Silwet L-77 to a concentration of 0.04% into the Arabidopsis infiltration culture just before ready for dipping. Mix
culture thoroughly.
8. Transfer Arabidopsis infiltration culture to a 300 mL beaker.
9. Prepare a stretch of plastic wrap ~20 cm  20 cm and spray it
with water.
10. Submerge Arabidopsis inflorescence completely for 30 s into
the infiltration media without exposing the leaves to the media.
Shake the pots while dipping into the media to make sure all
the flower buds are thoroughly exposed.
Biotin-Based Proximity Labeling of Protein Complexes in Planta
433