Editing Agrobacterium tumefaciens transformation (section)

==  Arabidopsis seed sterilization ==

For small amounts of seed (~10-500), work in 1.5 mL microfuge tubes. Note: 100 microliters of dry seed = ~2,500 seed

#To each tube, add 1 mL of 50% bleach/0.02% Tween 20, incubate with shaking/inversion for 7 minutes.
#Briefly centrifuge tubes to pellet seeds, remove bleach.
#Add 1 mL of sterile water and resuspend seeds to rinse off bleach. Spin briefly and remove wash.
#Repeat step 3 three more times (total of 4 rinses).
#Rususpend seed in ~100-200 uL of sterile 0.1% agarose and spread on growth media plates.

Suggestions: After the bleach is removed, you must work in a sterile hood. Work with sterile P1000 filter tips, freshly sterilized water and 0.1% agarose in wide-mouth bottles. BE CAREFUL not to touch the inner lip of microcentrifuge tube lids with anything nonsterile. For large amounts of seed (>2,500), work in 15 mL conical tubes.