Flagella and Axoneme Isolation

Chlamydomonas flagella and axonemes can be removed and purified using the following methods.

Flagella
Solutions:

Solutions for Isolation of Chlamydomonas Flagella make up the following fresh on day of use in disposable screw-top plastic tubes

DTT Stock
0.154 g in 10 ml H2O

0.01M Hepes
10 ml 0.1M (Stock) Hepes
90 ml H2O

HMDS enough for 1 x 250 ml culture
1 ml 0.1M Hepes
0.5 ml 0.1M MgSO4
ml stock DTT
0.4 g sucrose
8.4 ml H2O
Dibucaine
0.1 g in 10 ml H2O

HMDEgS 4% (enough for 2 x 250 ml cultures)
5 ml 0.1M Hepes
2.5 ml 0.1M MgSO4
0.5 ml stock DTT
250 ml 0.1M EGTA
2 g sucrose
41.75 ml H2O

HMDEgS 25% (enough for 2 x 250 ml cultures)
1 ml 0.1M Hepes
0.5 ml 0.1M MgSO4
0.1 ml stock DTT
50 ml 0.1M EGTA
2.5 g sucrose
8.35 ml H2O

HMDEdNa low
1 ml 0.1M Hepes
0.5 ml 0.1M MgSO4
0.1 ml stock DTT
10 ml 0.5M EDTA
0.0175 g NaCl
8.4 ml H2O

K-acetate (from Howard et al.)
1 ml 0.1M Hepes
0.5 ml 0.1M MgSO4
0.1 ml stock DTT
0.1 ml 0.1M EGTA
0.1 ml 5M K-acetate
8.2 ml H2O


Axonemes