Difference between revisions of "Team:Waterloo/Experiments"
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| − | + | Arabidopsis Transformation (Floral Dip) | |
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| + | <ul>Day0- Make Rif/Gm/km plates | ||
| + | <li>Day1- Electroporate your constructs into agrobacterium. Make sure you use appropriate antibiotic plates. For our agrobacterium it needs Rif and Gm and as long as we have pCAMBIA in it we need to add Km. Refer to the lab book with sticky notes.</li> | ||
| + | <li>Day2- Wait</li> | ||
| + | <li>Day3- Streak your plate if you dip on the 8th day. If you are in a rush just inoculate a single colony into 5mL of YEP media using appropriate antibiotic. You can dip in day 6 if you inoculate directly from the transformation plate. Incubate the tubes at 28 degrees. At this day please let me know so I can send an email to Charles lab regarding the change in incubation temperature. </li> | ||
| + | <li>Day4- <ol><li>Miniprep 1-2mL of your inoculated culture and run a pcr check+diagnostic gel. when minipreping agrobacterium make sure to wash 3-4 times.</li> | ||
| + | <li>Inoculate about 20ul from your last night culture into new 5mL of YEP</li> | ||
| + | <li>Make 1-2ml of YEP into 500ml baffled flask (depending on how many constructs you have. for example. One flask for pCAMBIA in agro, one for pCAMBIA+SgRNA, one for pCAMBIA+Cas9, one for pCAMBIA+Cas9+SgRNA-A2, one for Agrobacteriumalone) Fill up the flasks with 250mL of media and cover it with cap or sponge and foil. We have three flasks from Barb that have metal caps. Use the other flasks from Charles lab. Put the media in your flasks and autoclave them together.</li> | ||
| + | <li>Inoculate agrobacterium into 5mL YEP using only Rif and GM</li></ol> | ||
| + | |||
| + | <li>Day5- Take all your 5mL tubes that you inoculated overnight and all your ready flasks. Add your antibiotics to the flasks. When you have your constructs add all three Km/Gm/Rif, for your agrobacterium use only Rif/Gm. From your 5mL tube add 2mL of culture to the 250mL dilution. Do this at 4pm so you can start the dip at noon on the next day. Incubate the flasks at 28 degrees with shaker on. Refer to the lab book for antibiotic concentrations. Get all your solutions ready. Do not autoclave your sucrose, you can just make it on the day of the dip. Make sure that you make rest of the solutions though. Don’t remember the other solution but that one need to be made and autoclaved on this day. </li> | ||
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| + | <li>Day6- Dip Day! Get to the lab 1 hour before the dip. Turn on the big centrifuge make sure it is at 4 degrees.Then take your cultures and spin down for 15min at 4 degrees. Make your sucrose. Take your cultures Read the OD. Calculate your dilutions and amount of Silwet that you need to add. For resuspending, add 50ml of your solution, and pipette up and down to break the pellet. Then just have fun dipping! Make sure you autoclave the trays first. </li></ul> | ||
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Revision as of 01:13, 19 September 2015
Experiments & Protocols
- Seeds of Arabidopsis thaliana
- Agrobacterium tumefaciens strains such as C58C1
- Luria-Bertani (LB) media (broth and agar plate)
- Induction medium: 10.5 g/L K2HPO4, 1 g/L (NH4)2SO4, 0.5 g/L NaCitrate, 1 g/L glucose, 1 g/L fructose, 4 g/L glycerol, 1 mM MgSO4, 10 mM 2-(N-morpholino) methanesulfonic acid (MES); adjust pH to 5.6, autoclave before use
- Infiltration medium: 10 mM MgSO4, 10 mM MES; adjust pH to 5.6, autoclave before use
- Rifampicin: dissolve in methanol to make 25 mg/mL stock solution and store in -20 C freezer
- Tetracycline: dissolve in 70% ethanol to make 12.5 mg/mL stock solution and store in -20 C freezer
- Kanamycin: dissolve in water to make 50 mg/mL stock solution and store in -20 C freezer
- Acetosyringone (3,5-dimethoxy-4-hydroxyacetophenone, Aldrich): dissolve in 196.2 mg acetosyringone in 1 mL dimethylsulfoxide to make 1 M stock solution and store in -20 C freezer
- Dexamethasone (DEX, USB, Cleveland, OH): dissolve 78.4 mg DEX in 1 mL ethanol to make 200 mM stock solution
- 5-bromo-4-chloro-3-indolyl B-D-glucuronide (X-Glu, Sigma, St. Louis, MO): dissolve X-Glu in dimethylformamide to make 200 mM stock solution and store in -20 C freezer
- GUS staining solution: 50 mM sodium phosphate (pH 7.0), 1 mM EDTA, 2 mM X-Glu, 0.05% SDS, 0.1% sodium N-lauryl-sarcosine, 0.1% Triton-X-100
- Vacuum pump
- Day0- Make Rif/Gm/km plates
- Day1- Electroporate your constructs into agrobacterium. Make sure you use appropriate antibiotic plates. For our agrobacterium it needs Rif and Gm and as long as we have pCAMBIA in it we need to add Km. Refer to the lab book with sticky notes.
- Day2- Wait
- Day3- Streak your plate if you dip on the 8th day. If you are in a rush just inoculate a single colony into 5mL of YEP media using appropriate antibiotic. You can dip in day 6 if you inoculate directly from the transformation plate. Incubate the tubes at 28 degrees. At this day please let me know so I can send an email to Charles lab regarding the change in incubation temperature.
- Day4-
- Miniprep 1-2mL of your inoculated culture and run a pcr check+diagnostic gel. when minipreping agrobacterium make sure to wash 3-4 times.
- Inoculate about 20ul from your last night culture into new 5mL of YEP
- Make 1-2ml of YEP into 500ml baffled flask (depending on how many constructs you have. for example. One flask for pCAMBIA in agro, one for pCAMBIA+SgRNA, one for pCAMBIA+Cas9, one for pCAMBIA+Cas9+SgRNA-A2, one for Agrobacteriumalone) Fill up the flasks with 250mL of media and cover it with cap or sponge and foil. We have three flasks from Barb that have metal caps. Use the other flasks from Charles lab. Put the media in your flasks and autoclave them together.
- Inoculate agrobacterium into 5mL YEP using only Rif and GM
- Day5- Take all your 5mL tubes that you inoculated overnight and all your ready flasks. Add your antibiotics to the flasks. When you have your constructs add all three Km/Gm/Rif, for your agrobacterium use only Rif/Gm. From your 5mL tube add 2mL of culture to the 250mL dilution. Do this at 4pm so you can start the dip at noon on the next day. Incubate the flasks at 28 degrees with shaker on. Refer to the lab book for antibiotic concentrations. Get all your solutions ready. Do not autoclave your sucrose, you can just make it on the day of the dip. Make sure that you make rest of the solutions though. Don’t remember the other solution but that one need to be made and autoclaved on this day.
- Day6- Dip Day! Get to the lab 1 hour before the dip. Turn on the big centrifuge make sure it is at 4 degrees.Then take your cultures and spin down for 15min at 4 degrees. Make your sucrose. Take your cultures Read the OD. Calculate your dilutions and amount of Silwet that you need to add. For resuspending, add 50ml of your solution, and pipette up and down to break the pellet. Then just have fun dipping! Make sure you autoclave the trays first.
Describe the experiments, research and protocols you used in your iGEM project.
What should this page contain?
- Protocols
- Experiments
- Documentation of the development of your project