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Team:Washington/MAKING GLYCEROL FROZEN STOCKS
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<a href="https://docs.google.com/document/d/19IXxggALb9FxxdzAu4593g3quBs1JZgo1vpj2-hjtNQ/edit?usp=drive_web">Streaking a Plate for Isolation</a><br /> | <a href="https://docs.google.com/document/d/19IXxggALb9FxxdzAu4593g3quBs1JZgo1vpj2-hjtNQ/edit?usp=drive_web">Streaking a Plate for Isolation</a><br /> | ||
Pick only a single well isolated colony for making glycerol stocks. <br /> | Pick only a single well isolated colony for making glycerol stocks. <br /> | ||
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<strong>Summary:</strong> <br /> | <strong>Summary:</strong> <br /> | ||
For the long-term storage of microbial cultures <br /> | For the long-term storage of microbial cultures <br /> | ||
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1. Grow overnight culture (1 mL) of your cells in selective media <br /> | 1. Grow overnight culture (1 mL) of your cells in selective media <br /> | ||
2. Mix 500 mL of your culture with 500 mL of 40-60% glycerol in a cryo tube (or 1.5mL tube). Immediately place at <strong>-80 C</strong> (high concentration of glycerol is toxic to cells and prolonged exposure to it kills them at RT). <br /> | 2. Mix 500 mL of your culture with 500 mL of 40-60% glycerol in a cryo tube (or 1.5mL tube). Immediately place at <strong>-80 C</strong> (high concentration of glycerol is toxic to cells and prolonged exposure to it kills them at RT). <br /> | ||
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<p><strong>Making <em>E-coli</em> glycerol stock:</strong></p> | <p><strong>Making <em>E-coli</em> glycerol stock:</strong></p> | ||
<p><strong>Material needed:</strong></p> | <p><strong>Material needed:</strong></p> | ||
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<li>Mix briefly by vortexing and store at -80 C </li> | <li>Mix briefly by vortexing and store at -80 C </li> | ||
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<strong>Growing cells from glycerol stock:</strong> <br /> | <strong>Growing cells from glycerol stock:</strong> <br /> | ||
With sterile pipet tip, touch the surface of the glycerol stock (DO NOT THAW), and throw the tip in 5 ml LB media with appropriate antibiotics. </p> | With sterile pipet tip, touch the surface of the glycerol stock (DO NOT THAW), and throw the tip in 5 ml LB media with appropriate antibiotics. </p> | ||
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Revision as of 03:27, 21 September 2013
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Making Glycerol Stocks
Streaking a Plate for Isolation
Pick only a single well isolated colony for making glycerol stocks.
Summary:
For the long-term storage of microbial cultures
Make sure that you do not contaminate glycerol stocks and they do not thaw.
1. Grow overnight culture (1 mL) of your cells in selective media
2. Mix 500 mL of your culture with 500 mL of 40-60% glycerol in a cryo tube (or 1.5mL tube). Immediately place at -80 C (high concentration of glycerol is toxic to cells and prolonged exposure to it kills them at RT).
Making E-coli glycerol stock:
Material needed:
- Growing E-coli (mid log phase)
- 40%-60% glycerol stock (in water, autoclaved)
- Cryo-tubes
To make the E-coli stock:
- Inoculate 5 ml LB media using a single colony with appropriate antibiotics. Allow to grow to mid log phase(~12hrs).
- In cryo-tube add 500 ul of growing E-coli.
- Add 500 ul of 40%-60% autoclaved glycerol.
- Mix briefly by vortexing and store at -80 C
Growing cells from glycerol stock:
With sterile pipet tip, touch the surface of the glycerol stock (DO NOT THAW), and throw the tip in 5 ml LB media with appropriate antibiotics.
