Chlamydomonas Protocols and Recipes

TAP: Tris Acetate Phosphate Medium

Tris-Acetate Solution

150 mL Distilled Water
60.5 g Tris Base
20 mL Glacial Acetic Acid
titrate to pH 7.0 (likely 5 mL more needed)
bring to 250 mL total volume with distilled water

Macroelement Solution

150 mL Distilled Water
10 g NH4Cl *
2.5 g MgSO4·7H2O
1.25 g CaCl2·2H2O
bring to 250 mL total volume with distilled water

* Note: for nitrogen-free medium, replace with 13.9 g KCl

Phosphate Buffer

150 mL Distilled Water
2.7 g K2HPO4
1.4 g KH2PO4
should be pH 7.0
bring to 250 mL total volume with distilled water

Chelated Iron Solution

150 mL Distilled Water
4.2 g Sequestrene 330 (Fe:NaDTPA = C14H19FeN3O10Na, 468.15 g/mol)
bring to 250 mL total volume with distilled water

Microelement Solution

150 mL Distilled Water
0.715 g H3BO3
0.453 g MnCl2·4H2O
0.027 g ZnCl2
0.013 g CuCl2·2H2O
0.006 g CoCl2·6H2O
0.006 g Na2MoO4·2H2O
bring to 250 mL total volume with distilled water

Note: this combination of chelated iron and micronutrient solutions is modified from Murashige and Skoog (1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15(3): 473-497). It does not have the long preparation protocol of Hutner's. Due to the use of all-chloride salts and separation of Fe, both solutions are completely and stably soluble and do not require filtration of precipitates (i.e. you know what is left dissolved in these solutions!).

TAP Medium Preparation

800 mL Distilled Water
10 mL Tris-Acetate Solution
10 mL Macroelement Solution
10 mL Phosphate Buffer
1 mL Chelated Iron Solution
1 mL Microelement Solution
bring to 1 L total volume with distilled water

For solid medium, add 15 g agar per liter
Autoclave at 15 psi, 121°C, 15 min

Mating Chlamydomonas

Grow up + and – mating types separately in TAP liquid medium to mid-green (107 cells/mL)
Spin down gently (3000 G) for 3 minutes
Decant supernatants
Resuspend pellets in 10 mM HEPES pH 7.2 buffer
(1M HEPES is a 100x stock = 238.3 g/L HEPES titrated with KOH)
Starve overnight in light (25 μmol photons/m2/sec) with aeration
Mix equal cell concentrations
Should show good mating frequency within 1 hour

Transforming Chlamydomonas

Culture cells for two days in TAP liquid medium to mid-green
Digest 2μg plasmid with restriction endonuclease to linearize (ampr = Xmn-1) plasmid
Use appropriate buffer and BSA if needed, incubate 37°C for 1 hour
Spin down gently (3000 G) for 3 minutes
Discard supernatant
Resuspend pellet in Autolysis solution (sub-micron filtrate from large mating culture) for 45 min
Spin down 3000 G for 3 minutes
Discard supernatant, remove residue
Resuspend pellet in 50 mL TAP liquid medium to wash
Spin down 3000 G for 3 minutes
Discard supernatant, remove residue
Resuspend pellet in 2 mL TAP liquid medium to wash
Mix together:
300μg 0.5 mm glass beads (HCl acid washed, rinsed to pH6, baked 400°F for 2 hours) 300μL resuspended wall-less cells
100μL 20% Polyethylene glycol 6000 (filter sterilized)
20μL Linearized plasmid digest mix
Vortex 15-30 seconds at high speed
Pipette and spread in TAPS solid medium plates with selective antibiotic (50 mg/L zeocin)
Try 100 μL in one plate, 1 μL plus 99μL TAPS liquid in another