"
Team:University of Alberta/Transformation Protocol
From 2008.igem.org
(New page: Made Availible thanks to Dr. Enrico Scarpella<br> '''Day 1'''<br> '''1.'''Inoculate 5ml of LB + Hygromycin (Our Vector carries a chromosomal resistance to Hygromycin)+proper antibiotic for...) |
|||
| Line 1: | Line 1: | ||
Made Availible thanks to Dr. Enrico Scarpella<br> | Made Availible thanks to Dr. Enrico Scarpella<br> | ||
'''Day 1'''<br> | '''Day 1'''<br> | ||
| - | '''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 | + | '''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<br> |
| - | '''Day 2''' | + | '''Day 2'''<br> |
'''1.''' Pour the 5ml culture into 250ml LB + Hygromycin + Antibiotic in a 1L flask | '''1.''' Pour the 5ml culture into 250ml LB + Hygromycin + Antibiotic in a 1L flask | ||
| - | '''Day 3''' | + | '''Day 3'''<br> |
| - | '''1.''' Spin down the 250ml culture at 4000rpm for 15 min (at 4 degrees C), Divide each 250ml culture over two 250ml bottles | + | '''1.''' Spin down the 250ml culture at 4000rpm for 15 min (at 4 degrees C), Divide each 250ml culture over two 250ml bottles<br> |
| - | '''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 | + | '''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<br> |
| - | '''3.''' Add ca. 125ml of 5% sucrose solution to each of the bottles. Mix Gently | + | '''3.''' Add ca. 125ml of 5% sucrose solution to each of the bottles. Mix Gently<br> |
| - | '''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 | + | '''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<br> |
| - | '''5.''' Pour the two 125ml suspensions into a sterile suitable container. | + | '''5.''' Pour the two 125ml suspensions into a sterile suitable container.<br> |
| - | '''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) | + | '''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)<br> |
| - | '''7.''' Place the pots with the dipped plants horixontally in a tray layered with paper towels. Cover with a dome. Do Not Water | + | '''7.''' Place the pots with the dipped plants horixontally in a tray layered with paper towels. Cover with a dome. Do Not Water<br> |
'''Day4''' | '''Day4''' | ||
Revision as of 22:09, 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
