Difference between revisions of "Team:Toronto/Experiment-SOC Medium Protocol"

 
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Latest revision as of 06:58, 19 October 2016

SOC Medium Protocol · Benchling

SOC Medium Protocol

Introduction

SOC is a variant of the rich media SOB (super optimal broth) with catabolite repression. This means that glucose is supplemented in the media, allowing for optimal metabolic conditions for the bacteria. SOC increases the transformation efficiency of cells, as it provides ample nutrients to cells that have recently undergone stress as result of having been made competent. Accordingly, it will be used in bacterial transformation to stabilize the cells and to increase transformation yields. Since SOC is high in nutrients, it is more easily contaminated than LB or King's B media. SAFETY PRECAUTIONS SDS (safety data sheet): Refer to the SDS sheets for all listed materials before entering the lab. Be prepared to answer any questions regarding the information on these sheets. PPE (Personal protective equipment): Proper lab attire should be worn throughout the experiment: This means that upon entering the lab you should be wearing long pants and close-toed shoes. Contact lenses should not be worn. Furthermore, a lab coat, goggles, and gloves should be worn at all times, and long hair should be tied back. Autoclave: The autoclave should only be handled by execs. ***Note that any autoclaved materials may still be hot and should therefore be handled with caution. Be careful not to burn yourself.

Materials

  • Reagents
    • 1.802g glucose
    • 10g tryptone
    • 2.5g Yeast extract
    • 0.584g NaCl
    • 0.093g KCl
    • 1.016g MgCl2 (anhydrous)
    • 1.234g MgSO4.7H2O
    • 500ml of dH20 (distilled water)
  • Materials
    • 2 x 1L pyrex bottle (must have cap) ␥Note: A smaller pyrex bottle may be used to accommodate the glucose solution.
    • 1L graduated cylinder
    • Weighing boats and scupula
    • 70% EtOH wash bottle
    • Paper towels/wipes

Procedure

  • Part I: Making the SOC broth (this step can be carried out at a regular lab bench)
  1. Obtain two 1L mL pyrex bottles. Ensure that the bottles can be sealed with a cap - this will help prevent contamination and enable long term storage.
  1. Obtain a graduated cylinder with 500ml of dH20 (distilled water).
  1. Using filter paper, separately measure out 10g tryptone, 2.5g yeast extract, 0.292g NaCl, 0.093g KCl, 1.016g MgCl2 anhydrate, 1.234g MgSO4, heptahydrate. on a scale and add them to the 1L bottle.
  1. Add 400mL dH20. Swirl the flask in a circular motion to mix. Remember to recalibrate your scales in between measurements.
  1. In the separate bottle, and measure out 1.802g glucose on a scale and add the rest of the 100mL dH20. Recalibrate your scales in between measurements.
  • **** The 1L autoclaved separately, as the contents will react if autoclaved together. ***
  • Autoclaving **ONLY DONE BY EXECS**
  1. Lightly seal the top of the bottles with aluminium foil, or unscrew the caps. Label both with autoclave tape. Include a label with SOC – your name – date - iGEM 2015.
  • **Unless you have been trained to use the autoclave, you will not be conducting the following steps 2 - 5.
  1. Use appropriate transportation protocols to bring the bottles into the autoclave room. Remember to store the beaker in an autoclavable basin, in case of spills.
  1. Check the water level on the autoclave, if necessary. Autoclave on the liquid setting for approximately 20mins.
  1. ***The contents of the bottles will be hot after autoclaving, therefore take necessary measures to prevent burns. After autoclaving, allow the media too cool to 55oC before handling.
  • Part II: Making the SOC broth (this part should be done in room WB303, in sterile conditions close to a bunsen burner)
  1. In a sterile environment, slowly add the autoclaved 1.802g glucose and dH20 to the beaker containing the autoclaved 1.016g MgCl2 and 1.234g MgSO4, 10g tryptone, 2.5g yeast extract, 0.292g NaCl, 0.093g KCl and dH20. Flame the lip of the bottle before transferring the contents. Swirl to mix, and seal tightly to prevent contamination. Flame the cap before sealing.
  1. The SOC broth can be stored in sterile conditions at room temperature, and should be good for a 3-4 months. Flame the lip of the bottle each time the SOC is used. SOC should be handled carefully, as it is especially prone to contamination.
  • Before leaving the lab
  • ⠀Clean dirty glassware, or at least set aside the glassware to be cleaned by a designated individual. ⠀Wipe down your workspace.
  • ⠀Ensure that all materials have been returned to their places, and that the plates have been properly stored in the fridge.
  • References
  • Changelog
  • 1. Created 7/5/2016



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