Team:Washington/MAKING GLYCEROL FROZEN STOCKS

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Making Glycerol  Stocks<br />
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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 />
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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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3. LABEL VERY WELL and add information  to drive. <br />
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See Labeling Protocol: <br />
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<strong>Freezer Stocks (Glycerol Stocks)</strong>
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  <br />
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  Plasmid Name <br />
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  Backbone <br />
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  Antibiotic  Resistance, &nbsp;&nbsp;&nbsp;Cell Type <br />
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Initials of who  prepared the stock, &nbsp;&nbsp;&nbsp;Date</blockquote>
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If you need to take from the stocks, do  not thaw - scrape ice off of the top with a sterile loop and streak across  plate.
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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  &nbsp; </li>
   <li>Mix briefly by vortexing and store at -80 C  &nbsp; </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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Latest revision as of 04:35, 27 September 2013


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:

  1. Growing E-coli (mid log phase)
  2. 40%-60% glycerol stock (in water, autoclaved)
  3. Cryo-tubes

To make the E-coli stock:

  1. Inoculate 5 ml LB media using a single colony with appropriate antibiotics. Allow to grow to mid log phase(~12hrs).
  2. In cryo-tube add 500 ul of growing E-coli.
  3. Add 500 ul of 40%-60% autoclaved glycerol.
  4. 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.

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