Co-Cultivation Medium



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) |10ml |20ml |30ml |40 ml | |

|4°C fridge | | | | | |

|100x B5 Minor (1X final) |1.0ml |2.0ml |3.0ml |4.0ml | |

|4°C fridge | | | | | |

|100x FeSO4/NaEDTA |1.0ml |2.0ml |3.0ml |4.0ml | |

|(MSIII Iron) (1X final)) | | | | | |

|4°C fridge | | | | | |

|MES (20mM final) |3.9g |7.8g |11.7g |15.6g | |

|Cabinet (Sigma cat# M8250 (1kg); M2933 (100g)) | | | | | |

|Sucrose (3% final) |30g |60g |90g |120g | |

|Cabinet | | | | | |

| |

|pH 5.4 pH= |

|Agar (0.8% final) |5g |10g |15g |20g | |

|Cabinet (Sigma cat# A-1296) | | | | | |

|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-Cultivation add-ins

Prepared by:____________

Date:____________

|Ingredients |Amount of media |Actual |

| |1L |2L | |

|BAP (BenzylAminoPurine Sol.) (1.67mg/L final) |1.67ml |3.34mll | |

|4°C fridge (PhytoTechnology labs cat# B130-100mL) | | | |

|GA3 (Gibberellic acid) (0.25mg/L final) |0.25ml |0.50ml | |

|4°C fridge (Sigma cat# G-7645) | | | |

|100x B5 vitamins (1/10 strength final) |10ml |20ml | |

|4°C fridge | | | |

|DDT (1mM final) |0.154g |7.7mg | |

|4°C fridge | | | |

|Sodium Thiosulfate (1mM final) |0.248g |7.9mg | |

|Cabinet | | | |

|Cysteine (8.3mM final) |1g |200mg | |

|Cabinet | | | |

|Acetosyringone (AS) (200um final) |40mg |80mg | |

|4°C fridge | | | |

|DMSO |0.5ml |1.0ml | |

|“Flammables” cabinet under fume hood | | | |

*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.

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