Difference between revisions of "Team:MIT/Protocols"

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Protocols
 
Protocols
 
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<li> Add 100ul of 1M CaCl<sub>2</sub> (sterile)</li>
 
<li> Add 100ul of 1M CaCl<sub>2</sub> (sterile)</li>
 
<li> Adjust to 1000ml with distilled H<sub>2</sub>O</li></ol>
 
<li> Adjust to 1000ml with distilled H<sub>2</sub>O</li></ol>
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Stock Culture Preparation
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<p>C Hutch is slow to grow, so experiments will be much faster if large initial concentrations of C Hutch is used.  This is achieved by having a continual stock of C Hutch in the incubator which is used to provide cells for experiments
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</p><p>Protocol:
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</p><p>Stripette:
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<ul><li> 10ml of current stock</li>
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<li> 90ml of DSM3T media</li>
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<li> 5g of autoclaved filter paper strips</li></ul>
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<p>into a 500ml flask, and place in shaker.
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Revision as of 14:26, 16 September 2015


Protocols
C. Hutchinsonii Media

This is the media used for C. Hutch and co cultures with E. Coli. The media contains all the salts needed for both bacteria but not the carbon source (filter paper) due to the carbon source not being liquid. Cultures are made by adding the salt solution to a tube containing a piece of filter paper where the tube with the paper in it has been sterilized (usually by autoclave).

C Hutch ALONE in 1 L

Paper: 50 grams per liter of media, which is one 0.5g 9cm No. 1 filter paper disks per 100 mL.

Component Amount
KH2PO4 0.2 g
MgSO4*7H2O 0.5 g
KCl 0.5 g
NaNO3 0.5 g
FeSO4*7H2O 20 mg
K2HPO4 0.8 g
tryptone 1 g

Autoclave 15 min.

E Coli ALONE (M9) in 1 L

  1. Make 5x M9 salts
    • To make M9 Salts aliquot 800ml H2O and add
      • 64g Na2HPO4-7H2O
      • 15g KH2PO4
      • 2.5g NaCl
      • 5.0g NH4Cl
      • Stir until dissolved
      • Adjust to 1000ml with distilled H2O
      • Sterilize by autoclaving
  2. Measure ~700ml of distilled H2O (sterile)
  3. Add 200ml of M9 salts
  4. Add 2ml of 1M MgSO4 (sterile)
  5. Add 20 ml of 20% glucose (or other carbon source)
  6. Add 100ul of 1M CaCl2 (sterile)
  7. Adjust to 1000ml with distilled H2O
Stock Culture Preparation

C Hutch is slow to grow, so experiments will be much faster if large initial concentrations of C Hutch is used. This is achieved by having a continual stock of C Hutch in the incubator which is used to provide cells for experiments

Protocol:

Stripette:

  • 10ml of current stock
  • 90ml of DSM3T media
  • 5g of autoclaved filter paper strips

into a 500ml flask, and place in shaker.