- 1. Grow cells in SGII+NH4NO3 until they reach a density of 1-2 x 10^6/ml.
- 2. Spin down cells at moderate speed (5000 rpm for 5 min in a GSA type rotor).
- 3. Resuspend in 1/100 volume SGII+KNO3 and allow to shake at room temperature for 2-4 hours.
- 4. If desired, treat cells with gamete autolysin (prepared as described in The Chlamydomonas Sourcebook*) for approximately 45 minutes. I usually resuspend the cells in about 1/25 the original volume in autolysin. Spin and wash once with SGII+KNO3 before transformation. Optional: Test effectiveness of lysis by sensitivity to 0.05% NP-40. (Count duplicate samples +/- detergent in hemacytometer.)
Note: Several recent experiments suggest that autolysin treatment increases the transformation rate of cw-15 mutants.
- 5. Work in lots of a few tubes at a time. Add 300 microliters cells, 100 microliters 20% polyethylene glycol, 1-2 micrograms DNA (linearized DNA generally transforms somewhat more efficiently than supercoiled), and 300 mg sterile glass*** beads. Vortex for 15-30 seconds at top speed on a Fisher Vortex Genie 2 mixer; plate cells immediately on an SGII+KNO3 selective agar**. Depending on age and moisture of plates, parafilm immediately or after 24 hours; and put in the light.
Colonies should be visible in about 6 days.
* The protocol is followed except that the culture supernatant of the mated gametes is filtered through a 0.22-0.45 micrometer nitrocellulose filter. I don’t know how much of the activity (if any) is lost in this protocol, but the requirement for sterility to my mind outweighs any loss in this step. After the filtration, the supernatant is frozen in 50 ml lots at -20 deg C.
** Use washed agar to remove traces of ammonia and other impurities.
***Glass beads are approximately 0.5 mm in diameter and are available from Thomas. We wash them with acid, rinse them with water until the pH is neutral, dry them, and then weigh 300 mg into glass culture tubes and bake for approximately 2 hours at 400 deg F.