Difference between revisions of "Team:Freiburg/Notebook"
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<li class="level1"><div class="li"> inoculate 2ml + Xml to measure OD (personal preference) LB-Medium to OD600=0,1 from overnight culture </div> | <li class="level1"><div class="li"> inoculate 2ml + Xml to measure OD (personal preference) LB-Medium to OD600=0,1 from overnight culture </div> | ||
Revision as of 14:44, 23 August 2016
Sporulation
Checklist:
- Use the right Medium.
- Vortex cuvettes if you make a dilution
- Set Nanodrop to cuvettes
1. Overnight culture
- inoculate your culture in 5ml LB-Medium
- let them grow over night at 37°C, 200rpm
2. Exponential growth
- measure the OD600 of your overnight culture
- inoculate 2ml + Xml to measure OD (personal preference) LB-Medium to OD600=0,1 from overnight culture
- let the cells grow to an OD600=0,8 (37°C, 200rpm)
3. Sporulation
- centrifuge the 2 ml of cells at 13.000 rpm for 1 minute
- wash the pellet with PBS
- resuspend the pellet in 1 ml DSM (Difco Sporulation Medium)Culture medium
- let the cells grow (24h,37°C 200rpm)
4. Lysozym treatment
- dilute the cells 6:1 in lysozym solution (15mg/μl)
- incubate for 1 hour at room temperature
- wash 6 times with 1x PBS
5. Count the spores (Neubauer-counting chamber, usually dilute in BPS 1 to 100)
6. Make aliquots (around 100million spores per aliquot)
