Transformation Protocol for Arabidopsis: Difference between revisions
(New page: Transformation Protocol for Arabidopsis – Abbreviated Germinate seed in pots ↓ 4 weeks Streak bacteria onto YM/MinA ↓ 2-3 days 28°C Spray/dip bacterial suspension o...) |
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Transformation Protocol for Arabidopsis | '''Transformation Protocol for Arabidopsis''' | ||
Method modified from Clough and Bent (1988) | Method modified from Clough and Bent (1988) |
Revision as of 21:02, 19 April 2007
Transformation Protocol for Arabidopsis – Abbreviated
Germinate seed in pots
↓ 4 weeks
Streak bacteria onto YM/MinA
↓ 2-3 days 28°C
Spray/dip bacterial suspension onto plants
↓ 1 day in box
Let plants set seed
↓ 3-4 weeks
Plate seed to GM + selection
↓
Transfer transformed plants to the glasshouse
Transformation Protocol for Arabidopsis
Method modified from Clough and Bent (1988)
Plant material: • Thinly sow Arabidopsis thaliana cv C24 seed into soil. Grow at 20°C under 16h light until plants are beginning to flower. Cut off any seed pods that have formed prior to transformation.
Prepare bacteria: • Streak YM plus antibiotic plates with bacteria, or Minimal A plus antibiotic plates for Agrobacterium. • Incubate plates for 2-3 days at 28°C.
Transformation: • Measure about 20mL Infiltration Medium for each bacterial strain. • Scrape or wash bacteria from plate with sterile loop and suspend in 20mL of Infiltration media. • Adjust density to an OD600 0.9-1.0. • Dip flowers and unopened buds of Arabidopsis into the bacterial suspension, or alternatively the plants can be sprayed with bacterial suspension using a plant mister. • Cover plants to maintain humidity overnight. Grow at 20°C in light. • Repeat dipping/spraying after 7 days. • Collect seed, store at 4°C
Selection: • Weigh seed to estimate number (100 seed ≈ 21.8mg) • Sterilise for 2 mins in 70% (v/v) ethanol followed by 20 mins in 20% (v/v) bleach (Zixo brand) + 0.02% Triton X-100. Keep the seeds moving in the bleach solution by putting tubes on a rotator wheel. • Wash seeds 5 times with sterile distilled water (in laminar flow hood) • Plate to Germination Medium + selection plates, seal with Micropore tape. • Incubate overnight at 4°C then in light at 20°C. After 10 days select transformants, transplant to soil.
Media and solutions for Arabidopsis transformation
YM Media
1L
Mannitol 10g
Yeast extract 0.4g
K2HPO4 (10% w/v stock) 1 ml
KH2PO4 (10% w/v stock) 4 ml
NaCl (10% w/v stock) 1 ml
MgSO4.7H2O (10% w/v stock) 2 ml
• pH 6.8 • Agar 15g/L • Autoclave • When ready to pour add: • Antibiotic selection if required
Keep poured plates for 2 days at room temperature to visualise any contamination, then store at 4°C.
Minimal A Medium (Svab, et al, 1975)
Liquid 1L Stock solution A 490mL Stock solution C 11mL Milli-Q H2O 499mL
Solid 1L Stock solution A 490mL Stock solution B 499mL Stock solution C 11mL
• Store 4°C
Minimal A Stock Solution A
Final concentration g/490mL 1 x mM
K2HPO4 10.5 60 KH2PO4 4.5 33 (NH4)2SO4 1.0 7.6 tri-Sodium citrate.2H20 0.5 1.7
• Autoclave in 490mL aliquots • Store 4°C • Substituting chemicals • K2HPO4.3H2O 13.8g/490mL
Minimal A Stock Solution B
Dissolve 15g agar in Milli-Q H2O, make up to 500mL
• Autoclave • Store room temperature
Minimal A Stock Solution C
Add 1mL 1M MgSO4 to 10mL 40% sucrose
• Store room temperature
Infiltration media
200ml Final conc.
10X MS salts 20ml 1xMS salts Sucrose 10g 5% MES 0.2g 0.1% Silwet-L77 200