1/2X MS agar plates for Arabidopsis (Recipe for 1 liter)

1) To a 2 liter flask, add 2.2 g MS powder

2) Sucrose to 1 % final concentration (therefore, 10 g sucrose per liter)

3) 1 liter distilled water (from distilled water tap by sink)

4) add stir bar and mix on stir plate until everything is resuspended

5) pH to ~5.7 with 1 M KOH (check pH using pH meter in Cell lab; usually 1 small drop of 1M KOH will bring the pH up to 5.7)

6) add 7 g phytoblend agar (special agar for plants, do not use regular agar)

7) Cover top of flask with tin foil; Autoclave 20’; stir 30-60 minutes to cool (can place flask in 50 C water bath to cool- agar won’t solidify at this temp so you can hold at 50 C until ready to pour plates).

8) Add sterile antibiotics/herbicides (see table below) at this point, if required.

9) pour plates in hood in tissue culture room (make them thicker than usual so roots can grow better). Let plates dry with lids on for few hours to overnight. Put plates back in bag, tape shut, and label with type, date, and your name. Store in refrigerator, standing up.

Antibiotics/Herbicides

Chemical	Working concentrations (ug/ml)	Stock mg/ml	solvent
Kanamycin	75-2000	50	H2O
Hyg	10-400	10	H2O
Sulfa*#	7.5	7.5	H2O
BASTA	15** (for agar-grown), 120 (spray for soil-grown)	120	H2O

*Sodium-Sulfadiazine (mw = 272) Sigma S-6387
**make 15 mg/ml BASTA stock (=1000X for agar plates) in water and then filter sterilize, store in fridge.

#Sulfadiazine Foliar Application

For spray selection, seeds are sown in soil and germinated under a dome to maintain high humidity. Seedlings at the cotyledon stage are uncovered and sprayed to fully wet the plants and surrounding soil with a solution containing 0.03% L-77 silwet and sulfadiazine at concentrations of 500 mg/L. Following spraying, the seedlings are re-covered for 24-48 hours with a plastic dome to maintain high humidity. The sulfadiazine spray is reapplied similarly every third day for a total of four applications.

Arabidopsis Seed Sterilization Protocol

You will need:

• Seed wash solution (70% ethanol + 0.05% Triton X-100)
• 95% ethanol
• Whatman filter paper (circles)
• Sterile 1.5 ml microfuge tubes
• A sterile hood (Use right hand hood in cell culture room; make hood sterile by wiping down with ethanol from squirt bottle – usually a bottle of ethanol is close by. If hood beeps, move glass door down until it stops beeping)
• P-1000 pipetman and blue pipet tips
• Tweezers
• Bunsen burner and striker (should be one already in hood).
• Pencil
• sterile technique is a must; wear gloves and ethanol wash them often.  Watch what you touch. If you touch something outside the hood, like your nose, rinse  your gloves with ethanol.

1. Follow instruction on hood to open. Wipe down inside of hood with copious amount of 70% ethanol and/or bleach. Wipe down pipetman and anything else (racks, waste jar) with kimwipe soaked in ethanol.
   
   
2. Pour seeds (up to ~200 ul in each tube) in labeled microfuge tubes (best to use ethanol-resistant marker).

3. Working in a sterile hood, add 1 ml seed wash solution (70% ethanol + 0.05% Triton X-100) to seeds. Mix by hand or vortex occasionally for 5 min. Let the seeds sink to the bottom of the tube and remove the wash solution with pipetman.

4. Replace the first wash with 1 ml of 95% ethanol. Mix and let stand five minutes mixing occasionally.

5. Repeat step 3. Prepare filter papers during this step.

6. Prepare sterile filters by folding in half to make a crease, labeling with pencil, and soaking in 100% ethanol and allowing them to dry on the surface of the sterile hood.

7. Using a 1 ml pipette tip, transfer the seeds in a little bit of ethanol onto the filter paper and wait for the filters to dry.

8. When the seeds have dried, carefully pick up filter like a taco and sprinkle seeds onto plates by tapping on side of the filter paper. Try to spread seeds out on the plate, ~25 seeds per plate. Seeds can be arranged on the plate using a sterile tweezers (wet with ethanol and then flame) or pipet tip, which isn’t easy to do. Leftover sterile, dry seeds can be poured into a sterile microfuge tube for later use; label and date the tube. Seal plates with parafilm or surgical tape. Cold treat seeds (2-4 days) in refrigerator to help synchronize germination times. When the plates are put into a growth chamber place the agar side down so the roots grow down. For root assays, place plates on the vertical so roots grow on top of agar. If excess condensation appears in a plate, remove tape, open lid and give a quick flick to remove water; limit open time to reduce contamination; retape the plate.
   
   
9. Clean up hood, wiping down with ethanol, close, and turn off.
   Seedlings should germinate in ~3 days after placing in growth chamber, and antibiotic/herbicide resistance will be evident in ~10 days – non-transgenic seedlings will turn white and die. Note germination time, rate, and growth in your notebook.