Clough Lab

June 6, 2006

Co-Cultivation Medium (CCM)

Prepared by:______

Date:______

Ingredients / Volume / Actual amt added
1 L / 2 L / 3 L / 4 L
10x B5 Major (1X final)
4°C fridge / 10ml / 20ml / 30ml / 40 ml
100x B5 Minor (1X final)
4°C fridge / 1.0ml / 2.0ml / 3.0ml / 4.0ml
100x FeSO4/NaEDTA
(MSIII Iron) (1X final))
4°C fridge / 1.0ml / 2.0ml / 3.0ml / 4.0ml
MES (20mM final)
Cabinet (Sigma cat# M8250 (1kg); M2933 (100g)) / 3.9g / 7.8g / 11.7g / 15.6g
Sucrose (3% final)
Cabinet / 30g / 60g / 90g / 120g
pH 5.4 pH=
Agar (0.8% final)
Cabinet (Sigma cat# A-1296) / 5g / 10g / 15g / 20g
Final volume / 980ml / 1960ml / 2940ml / 3920ml

Wear protective gloves

Mixing Instructions

  • In an appropriate beaker, add B5 Major, B5 Minor, FeSO4/NaEDTA (also called MSIII Iron).
  • Place stirring rod in beaker and turn on stirrer.
  • Fill up beaker to half of final volume with ddH2O. Add MES and sucrose. Bring solution to appropriate final volume using a measuring cylinder.
  • Using pH meter: Remove indicator rod from storage solution and calibrate. Place beaker on platform. Turn on stirrer. Rinse of indicator rod with ddH2O into a different beaker, pat dry with Kimwipe, and remove small plug (if applicable) before placing in mixture.
  • Adjust pH to 5.4.
  • Turn off stirrer. Rinse rod with ddH2O and pat dry. Store in buffer solution.
  • Transfer the solution into appropriate sized bottles. Add agarose.
  • Microwave bottles in 5 minute increments until media is clear (no agarose particles visible)
  • Autoclave for 20 minutes (make sure cap is loose!)
  • Place hot medium in 50C water bath for at least 20 minutes to cool.
  • When the bottle is cool enough to handle, add CCM add-ins into solution
  • Use 100x15ml Petri dishes. (thin dishes)

Co-Cultivationadd-ins

Prepared by:______

Date:______

Ingredients / Amount of media / Actual
1L / 2L
BAP (BenzylAminoPurine Sol.) (1.67mg/L final)
4°C fridge (PhytoTechnology labs cat#B130-100mL) / 1.67ml / 3.34mll
GA3 (Gibberellic acid) (0.25mg/L final)
4°C fridge (Sigma cat# G-7645) / 0.25ml / 0.50ml
100x B5 vitamins (1/10 strength final)
4°C fridge / 10ml / 20ml
DDT (1mM final)
4°C fridge / 0.154g / 7.7mg
Sodium Thiosulfate (1mM final)
Cabinet / 0.248g / 7.9mg
Cysteine (8.3mM final)
Cabinet / 1g / 200mg
Acetosyringone (AS) (200um final)
4°C fridge / 40mg / 80mg
DMSO
“Flammables” cabinet under fume hood / 0.5ml / 1.0ml

*Prepared by diluting B5 vitamins (Gamborg’s Vitamin solution – Sigma cat# G1019) from 4°C fridge

Mixing Instructions

  • Prepare a 1.5ml microfuge tube with appropriate amount of DMSO (located in the “Flammables” cabinet under fume hood).
  • Use analytical balance to measure Cysteine and Acetosyringone samples.
  • Add Acetosyringone to the DMSO. Vortex and check if powder is dissolved; if not, place tube in water bath (42°C) for about 30 seconds and vortex again. Do a quick spin in the centrifuge and add the entire amount to the beaker.
  • Add remaining ingredients directly to autoclaved media. Mix well before pouring plates.