Team:Bilkent UNAM Turkey/Protocols
From 2011.igem.org
Transformation of
Chlamydomonas with Glass Beads
contributed by Karen Kindle
- 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.