Preparation of Autolysin
Based on the protocol in the lab methods notebook
- Toothpick a single colony and grow up in 0.2 ml TAP or M medium for 2-3 days until culture becomes moderately green. Transfer the 0.2 ml culture to 10 ml of TAP medium in a glass tube, grow under bright light for 3-4 days until dark green. Spread 1.5 ml of this culture on a large plate of M agar. Make 2 plates for each mating-type strain. Incubate the plates under bright light for 4-5 days until a green lawn of cells is formed on each plate.
- Add 15 ml of M-N medium to each plate, scrape off cells by gently spreading with a bent glass pipette. Transfer cells to a sterile 50 ml tube.Wash the plate once with another 10 ml of M-N. Combine cells from the two plates of each strain.
- Pipette 20 µl of the cell suspension into 930 µl ddH2O, add 50 µl of 10% glutaraldehyde. Count the cell density with a hemacytometer.
(# counted) x (dilution factor) x (104) = cells/ml
- Centrifuge in IEC centrifuge for 10 mins at 2200 rpm. Pour off supernatant as quickly as possible after centrifugation is done.
- Resuspend cells in M-N to a final concentration of 8-10 x 107 cells/ml. Transfer to a sterile 500 ml flask and incubate culture under bright light for 3-4 hr.
- After 3-4 hr, mix a small sample of the two mating-type cultures and assess mating activity. If very good mating occurs, mix equal volume of the mating cultures in a sterile 1-liter flask. Incubate under bright light.
- After 30 mins, fix a small sample of the mating mixture with glutaraldehyde. Estimate the percentage of quadriflagellates. If it is greater than 80%, go to the next step, else repeat steps 1-6.
- Transfer mating mixture to a sterile 250 ml round bottom bottle, centrifuge at 2200 rpm for 15 mins, at 15 °C in the IEC centrifuge.
- Transfer supernatant to a sterile container. Aliquot 5-10 ml fractions and store in -80 °C.
- Immediately prior to use filter the autolysin through a 0.45 µm filter. This will filter out any chlamy that stayed with the autolysin and get rid of any inadvertant microbial contamination.
- Grow up 200 ml of cells in a flask. Count the cell number and transfer 5 x 107 cells to each 15 ml corning tube. Centrifuge at top speed in a clinical table-top centrifuge. Discard supernatant.
- Thaw autolysin at room temperature. Add 0.5, 1, 2, or 4 ml of autolysin to cell pellets. Resuspend cells by gently shaking the tubes. For control, use M-N to resuspend cells. Incubate at room temperature with gentle shaking for 45 mins.
- After incubation, add 50 ml of each sample to 50 ml of 0.5 % glutaraldehyde or 1% NP-40 in a microtiter well. Estimate % of lysis as soon as possible (within 5 mins after addition to NP-40). For use in transformation, an autolysin prep that gives more than 80% cell lysis will be required.
This page is, as always, Copyright (c) 1997 by Craig D. Amundsen. All rights reserved.