1. Seeds of Arabidopsis thaliana
2. Agrobacterium tumefaciens strains such as C58C1
3. Luria-Bertani (LB) media (broth and agar plate)
4. 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
5. Infiltration medium: 10 mM MgSO4, 10 mM MES; adjust pH to 5.6, autoclave before use
6. Rifampicin: dissolve in methanol to make 25 mg/mL stock solution and store in -20 C freezer
7. Tetracycline: dissolve in 70% ethanol to make 12.5 mg/mL stock solution and store in -20 C freezer
8. Kanamycin: dissolve in water to make 50 mg/mL stock solution and store in -20 C freezer
9. 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
10. Dexamethasone (DEX, USB, Cleveland, OH): dissolve 78.4 mg DEX in 1 mL ethanol to make 200 mM stock solution
11. 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
12. 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
13. 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-
  1. Miniprep 1-2mL of your inoculated culture and run a pcr check+diagnostic gel. when minipreping agrobacterium make sure to wash 3-4 times.
  2. Inoculate about 20ul from your last night culture into new 5mL of YEP
  3. 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.
  4. 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.

Day 1:

1. Set-up an aseptic work area using either a bunsen burner or flow-hood. Use 70% ethanol to clean the bench top.
2. Use a marker to circle all the colonies you want to screen on the transformation plate(s), and label each one with a number.
3. Streak purify each of these colonies on LB agar plates with the corresponding antibiotic. Number these plates according to the colonies they came from.
4. Seal the plate’s edge with parafilm and incubate 37C overnight (at least 12hrs)

Day 2:

1. Set-up an aseptic work area using either a bunsen burner or flow-hood. Use 70% ethanol to clean the bench top. Remove plates from the incubator.
2. Based on the number of colonies you want to screen, use a permanent marker to draw multiple ~1 cm diameter circles on the LB agar plate. This is the “patch plate.” If you are screening too many colonies to fit on one patch plate, make a second patch plate. Label the circles on the patch plates with numbers that will correspond to the colony they came from.
3. Add 20uL of the Cell Lysis Solution to a 1.5mL microfuge tube. Repeat this step for however many colonies you want to screen.
4. Scoop up one selected colony from each of the streak purification plates using an inoculation stick.
5. Mini-streak the colony into a patch plate circle with the corresponding number.
6. Dip the same stick into the Cell Lysis Solution and swirl stick to get all bacteria off the stick.Discard stick into waste jar for autoclaving.
7. Repeat steps 7-10 for all colonies to be screened.
8. Ensure all the caps of the tubes are closed and vortex quickly to resuspend cells.
9. Place tubes into an 80C water bath (heat block containing water, or water bath) and let them sit for at least 15 minutes.
10. You may also choose to microwave your tubes, caps open, for 30 seconds to guarantee all cells have lysed.
11. Vortex tubes again to resuspend cells, and centrifuge at 13,300RPM for 4-5 minutes to pellet the cellular debris. Remove from centrifuge when complete.
12. Use the following recipe to prepare the colonies’ DNA for cPCR: Cell Template 2uL Forward Primer 1.25uL Reverse Primer 1.25uL Q5 HF Master Mix 10uL PCR MilliQ Water 5.5uL TOTAL VOLUME 20uL Repeat this for all colonies to be screened. **The primers should be designed to amplify your insert. Remember to do a positive AND negative control
13. Use the following PCR program to amplify the insert. First Cycle: 98C for 30s Second Cycle: 98C for 10-15s 68C* for 25-35s 72C for [30s/kb] Third Cycle: 72C for 3-5m 4C Forever and ever.
14. Upon completion of cPCR, run the samples in a diagnostic 1.0% agarose gel. Load 1uL of cPCR product, 4uL of de-ionized water, and 1uL of 6X Loading Dye.
15. Image gel and cry for happiness or sadness.

1. Pipette 100-600 ng plasmid into new tube and resuspend in 5uL dH2O, leave on ice
2. Take Agrobacterium electro competent cells and thaw 40uL (1 aliquot per transformation) on ice
3. Put cuvettes on ice before use with electroporator
4. Turn on electroporator, and set voltage at 1.25V, then press the CONST button
5. Pipette the 5uL plasmid mixture into the 40uL of competent cells. Mix by pipetting up and down a couple times, then transfer to cuvette
6. Wipe cuvette with a kimwipe, then transfer to electroporation unit. Press both buttons on the electroporator simultaneously and hold them down until you hear a beep and see a constant time on the screen. Record this value when finished (should be 3.7-4 ideally). Note: Lower values than 3.7 mean the cells were not washed with water sufficiently. If you see a spark or arc it means you have salt in your sample and need to precipitate and rinse it well with 70% ethanol.
7. As quickly as possible, at 1mL of YEP media to the cuvette and gently aspirate up and down to mix gently. Transfer to labelled growth tube.
8. Incubate at 27C for 2 hours
9. Gently mix the culture, pipette 20uL of culture and spreak on YEP agar with appropriate antibiotics and incubate at 28C (or room temp) for 3 days for colony to grow Note: These should also be in the dark.

1. Harvest 20 well-expanded leaves from 3- to 4-week-old plants before flowering
2. Cut 0.5 to 1 mm leaf strips from the middle part of a leaf on a clean white paper using a fresh, sharp razor blade without tissue crushing at the cutting site. Change the blade after cutting 4-5 leaves.
3. Transfer the leaf strips to 10 mL of enzyme buffer in a Petri plate by dipping both sides of the strips using a pair of flat-tip foreceps
4. Vacuum infiltrate the leaf strips for 10-15 minutes in the dark using a desiccator
5. Continue digestion at room temperature in the dark for >3 hours without shaking. The enzyme solution should turn green after a gentle swirling motion.
6. Check the release of protoplasts in the solution under a microscope (size = 30-50 um). Check the healthiness of protoplasts (unhealthy protoplasts show chloroplast clumping, irregularity in shape, and sometimes plasmolysis).
7. Filter the enzyme/protoplast solution through a piece of 75-um nylon mesh into a 50-mL tube.
8. Divide the protoplasts into two 15 mL tubes and pellet the protoplasts by centrifugation at 100 g (i.e. ~700 rpm) for 2 minutes using the swinging-bucket centrifuge H-20
9. (Optional) To remove unhealthy protoplasts and cell debris, resuspend the protoplast pellets in 10 mL of CS-Sucrose buffer and float the healthy protoplasts by centrifugation at 100 g for 2 minutes using the swinging-bucket centrifuge H-20. Remove the “internatant” and pellet as much as possible without disturbing the floating layer of healthy protoplasts.
10. Resuspend healthy protoplasts in 1 mL WI buffer and count cells using haemocytometer.
11. Resuspend the pellet (or floating layer of healthy protoplasts in step 10) in 1 mL of W5 solution by gentle swirling. Incubate on ice for 30 minutes. In the meantime, check the cell number using a haemocytometer.
12. The protoplasts should have settled to the bottom. Remove the supernatant and resuspend the pellet in Mg-Man buffer to get a concentration of 200 000 protoplasts per mL. Keep at room temperature and check the cell number again.
13. Add 5-10 ug of plasmid DNA in 10 uL to a 2 mL tube.

1. Grow seed culture to saturation (36-48 hours), need approximately 5mL of this
2. Inoculate 500mL of YEP media at a dilution of 1:100 Agrobacterium, and grow culture to an OD600 of 1.5 (takes approximately 24 hours)
Note: The rest of the protocol should be completed on ice
3. Spin cells at 5000 rpm for 15 minutes, resuspend in 500 mL cold, sterile, H2O
4. Spin cells at 5000 rpm for 15 minutes, resuspend in 250 mL cold, sterile, H2O
5. Spin cells at 5000 rpm for 15 minutes, resuspend in 10 mL sterile 10% (v/v) glycerol
6. Spin cells at 6000 rpm for 15 minutes, resuspend (wash) in 0.5 mL cold, sterile 10% (v/v) glycerol. Pipette the glycerol onto the cells and gently stir until well mixed. Cells will be viscous, and the final volume will be 0.003X original culture volume.
7. Dispense cells in 40uL aliquots into screw top tubes - sample is viscous so try to use a P1000 and it will make pipetting quicker and less difficult
8. Store at -80C and thaw on ice before use