Team:UGA/Experiments
Experiments
The following procedures were used to transform four conditions of plasmid into Nicotiana benthamiana.
Plasmids are as follows:
pJAB1489 (VP-16 driven by CaMV 35S promoter)
PJAB 1504 (6X UAS enhancing a minimal CaMV 35S promoter)
Gal1-BS3 (Gal1 promoter driving BS3 in PGWB1 backbone)
Condition 1: pJAB 1504+pJAB1489
Condition 2: Gal1-BS3
Condition 3: Agrobacterium EHA105 only
Condition 4: Induction buffer only
Transformation of EHA105 Agrobacterium
1. Inoculate 2.0 mL of LB with desired colony of Agrobacterium, incubate at 28°C overnight2. Cool on ice for 10 min
3. Pellet at 4°C at 1500xg for 5 min
4. Resuspend in 100μL of cold, sterile 20mM CaCl2
5. Add 2.0 μL of purified plasmid (about 1μg of DNA)
6. Freeze in liquid nitrogen
7. Incubate at 37°C for 4 min
8. Dilute in 1 mL of LB media and recover at 28°C shaking at 200 rpm for 4 hours
9. Pellet and spread on LB with selective media (rifampicin and kanamycin). Incubate at 28°C for 36-48 hrs
Agrobacterium Transient Expression
1. Grow 1mL overnight culture of Agrobacterium containing desired expression vector in LB+ appropriate antibiotic2. Pellet 0.2mL of overnight culture at 4000xg for 5 min
3. Decant media and resuspend pellet in 0.5mL of induction buffer or sterile water
4. Measure cell density at OD600
5. Dilute cell density to OD600 = 0.05 with induction buffer or sterile water
6. Hand infiltrate N. benthamiana leaves with 0.5mL of bacterial suspension using a 1mL blunt syringe
7. Check GFP expression at 2 and 4 days, and BS3 expression at 4 days.
Induction Buffer Recipe
39.45mL sterile water5mL sterile 500mM MES
5mL sterile 5% glucose
0.5mL sterile NaH2PO4
50μL sterile 200mM Acetosyringone