Arabidopsis transformation protocol (floral dip)

1.       Take 500 uL glycerol stocks and put into 4.5 ml 523 media with antibiotic. 25C, 200 rpm O/N.

2.       Inoculate with the whole 5 ml of agro into 200 ml of lb with antibiotics, 25C 200 rpm (4-5 hrs). Grow to an OD of 1-1.5.

3.       Spin down at 6000 rpm for 6 min. Decant and resuspend in:

a.      ½ Ms salt (2.15g/L)-0.654 g for 300 ml

b.      5% sucrose- 15 g for 300 ml

c.      Silwet 77 (90 ul/300 ml)

d.      Bring up to 300ml with water

4.       Dip flowers into solution for 5 seconds

5.       Cover the plants using a plastic dome lid and black cloth/trash bag

6.       Do not leave cloth for more than 16 hr

7.       To increase efficiency repeat procedure one week later. This depends on the stage of the flowers. A flower that has already produced a silique has already made the seeds and therefore will not be transformed by Agrobacterium

8.        Once silique turns brow-yellow (dried out) you can harvest the seeds. This takes about 1-2 weeks but can be sped up by not watering the plants.

Screening seeds:

Screening for positive plants:

1.       Bleach the T0 Seeds

• 10% bleach/water soln
• Add 1 mL, shake in epitube for 8-10 minutes
• Rinse with water 5 times

2.       Plate on ½ MS no sugar + 0.7 % agar +antibiotic medium (kan 50 mg/L)

• Use 1mL pipette to place seeds in rows on plate (see diagram)
• Top row will be purely wild-type seeds as a reference.

3.       Put in 4 C for 3-5 days

4.       Incubate at 20 C for several days, select that seedling with open and green cotyledon and root development.

5.       Transfer to soil/GA7