Easy single colony isolation of vegetative cells_28018326

---

title: "TheNessLab : Easy single colony isolation of vegetative cells"

1. TheNessLab
2. The Ness Lab Wiki
3. Chlamydomonas information and protocols

TheNessLab : Easy single colony isolation of vegetative cells

Created by Wen Chen on Feb 11, 2019

*Fool-proof and you get lots of single colonies. *

Procedure:

1. Inoculate strain of interest from working slant to 200 uL TAP in 96 well plate using an inoculating loop.
2. Incubate under light (25C, 100 rpm) for 2 days.
3. Before the dilution, mix the culture well with a pipette. 
4. Dilute the strain by adding 10 uL culture from 96 well plate to 990 uL TAP in a new plate. 
5. Transfer 10 uL from the diluted culture to to the center of a 1.5% agar in TAP plate. 
6. Spread the inoculum evenly following the general pattern below. Start with several horizontal streaks in the middle (dark blue), followed by slightly lighter blue streaks, and finish with the lightest blue streaks. 
   
7. Incubate under light (25C, any rpm) for 4-5 days until you have the ideal single colony sizes. You should end up with at least some single colonies near the edges of the agar plate.
8. Seal the plate with Parafilm. Use within one month. Some strains don't do well after one month in a sealed agar plates (most notably GB-117). 

Attachments:

[image2019-2-11_17-59-43.png](attachments/28018326/28018325.png) (image/png)

Document generated by Confluence on May 22, 2024 11:44

Atlassian