In article <199506301825.LAA02687 at net.bio.net>, S.Purton at ucl.ac.uk (Saul
Purton) wrote:
> We have had several goes at making autolysin according to the method
> described in the Sourcebook. However, our results have been rather
> disappointing with very little lytic activity as judged by the
sensitivity
> of pre-treated walled cells to NP-40.
>> Are we just incompetent or are there any secret tips for making good
preps?
Hi, Saul
By my experience, yield of the lytic activity is completely dependent
with the efficiency of mating. To get good mating, I am using good
mating strains, i.e., CC620 and CC621, both of them are available form
the Chlamydomonas Genetic Center. You might also be very careful for the
concentration of the cells during mating. If the concentration during
mating is too high, the efficiency of mating seems to become worse
(probably anaerobic environment is harmful).
The following is my protocol.
- Cultivate both mating type of the cells up to about 3x10^6 /mL in 250
mL of TAP in 1 L flask.
- Collect the cells by centrifugation at 3000 rpm 5 min.
- Resuspend the cells in 1L of TAP-N (NH4Cl was replaced with the same
concentration of KCl) in 2 L flask.
- Induce gamete by gently shaking for 24 hr under light.
- Harvest the cells by centrifugation at 3000 rpm 5 min.
- Resuspend each cells in 200 mL of TAP-N.
- Mix both mating type of cells in 2 L flask (larger vessel seems
better).
- Keep it under light without shaking for 1 to 2 hr to allow mating
(vigorous shaking seems harmful).
- Remove the cells by centrifugation at 8000 rpm.
- Remove cell debris by centrifugation at max speed.
- Filtrate the medium by 0.45 um membrane filter unit.
- Keep in -80C until use.
Kosuke Shimogawara
Department of Plant Biology
Carnegie Institution of Washington
p.s. Thank you very much for your cosmid libraries. It seems to work
well!
--
Kosuke Shimogawara@(kosuke at andrew.stanford.edu)
Carnegie Institution of Washington (U.S.A.)
Department of Plant Biology
