User:Burak Yilmaz/Notebook/YR10 protein interaction/2009/09/16

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E.coli Glycerol stock preperation

  • Making E-coli glycerol stock:

Material needed:

  • 1- Growing E-coli (mid log phase)
  • 2- 80% glycerol stock (in water, autoclaved)

To make the E-coli stock:

  • 1- Inoculate 5 ml LB media using a single colony. Allow to grow to mid log phase. Do not allow to saturate.
  • 2- In sterile tube (o-ring tubes), add 800 ul of growing E-coli.
  • 3- Add 200 ul of 80% 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.

alternative protocol

  • This protocol describes a method for storing bacterial cultures containing glycerol which are growing in liquid media. Based on CSH Protocols; 2006; doi:10.1101/pdb.prot4452



   * Bacterial culture growing in appropriate liquid medium
   * Glycerol, 60%, sterile (see Step 1)
   * nutrient (LB, TSB, etc.) agar plate containing the appropriate antibiotic 


   * Incubator, preset to correct temperature for growth
   * Inoculating loop, sterile/sterile toothpicks/sterile glass capillary tube
   * Tube, storage, with screw cap and air-tight gasket 


1. Sterilize glycerol by autoclaving for 20 minutes at 15 pounds per square inch (psi) (1.05 kg/cm2) on liquid cycle.

2. To 1.5 ml of bacterial culture, add 0.5 ml of the sterile glycerol in a labeled storage tube (final glycerol concentration of 15%). Label the top with a strain ID and the side with more detail (strain, date, initials).

3. Vortex the culture to ensure that the glycerol is evenly dispersed.

4. Optional: Freeze the culture in ethanol-dry ice or in liquid nitrogen.

5. Transfer the tube to the -80°C freezer, and record its location and contents in your notebook.

6. To recover the bacteria, scrape the frozen surface of the culture with a sterile inoculating loop, and then immediately streak the bacteria that adhere to the needle onto the surface of an LB agar plate containing the appropriate antibiotic. Return the frozen culture to storage at -80°C. Incubate the plate overnight to get individual colonies.


   * Best results are probably had from liquid cultures that are in late exponential growth, but stationary phase overnight cultures are usually also fine.
   * Some antibiotics can cause problems in long-term storage. Spin down the culture and resuspend in plain medium if you are concerned about this.
   * Some people use higher glycerol concentrations (25%,50%) for their stocks. Higher percentages may be good for longer-term storage, but this is speculation. Sean 15:57, 20 September 2007 (EDT)
   * You can also aliquot glycerol into screw-cap tubes and autoclave for 15 minutes. 

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