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From 2008.igem.org

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Revision as of 22:06, 27 May 2008

Made Availible thanks to Dr. Enrico Scarpella
Day 1
1.Inoculate 5ml of LB + Hygromycin (Our Vector carries a chromosomal resistance to Hygromycin)+proper antibiotic for construct selection in a 125ml flask with the UofA vector Agrobacterium strain harbouring the construct of interest. Typically, Scrape off a bit of frozen (-80 degrees C) glycerol stock of the strain with a yellow tip, and then use the tip as the inoculum. Grow at 28-30 degrees C with 200-250 rpm shaking

Day 2 1. Pour the 5ml culture into 250ml LB + Hygromycin + Antibiotic in a 1L flask

Day 3 1. Spin down the 250ml culture at 4000rpm for 15 min (at 4 degrees C), Divide each 250ml culture over two 250ml bottles 2. Pour off supernatant. Resuspend pellet in a few (3-4)ml of 5% sucrose (in demi water; freshly prepared) by gently pipetting up and down with a disposable platic pipette 3. Add ca. 125ml of 5% sucrose solution to each of the bottles. Mix Gently 4. Just before dipping the plant add 6.5 microliters of Silwet L-77 to each of the bottles (final conc. 0.05%). Mix Gently 5. Pour the two 125ml suspensions into a sterile suitable container. 6. Dip inflorescences into the solution for about 10-20 seconds while gently shaking. Use pots with 4-5 plants and 3-4 pots per construct( minimum 15 plants per construct) 7. Place the pots with the dipped plants horixontally in a tray layered with paper towels. Cover with a dome. Do Not Water

Day4 Turn pots with dipped plants upright. Water Them

Repeat the whole procedure (Day 1-4) after one week

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