Team:University of Chicago/Notebook/Media

From 2008.igem.org

Contents

Media

LB Broth (1 L)

  • 1 L dH20
  • 10 g Bacto tryptone
  • 5 g Bacto yeast extract
  • 5 g NaCl
  • 1 mL 2N NaOH
  • 12 g agar (optional)
    • 3 g agar + 250 mL broth per bottle
  • Autoclave

Note: Add 3 mL 1 M CaCl2 before phage transductions

PYE

This is a general-purpose Caulobacter medium and is typically used for plates and starter cultures from the freezer and for colonies off plates. Plates are incubated at 30C. Caulobacter colonies typically take 2-3 days to appear.

Per liter,

  • 2g bacto-peptone
  • 1g yeast extract
  • 200mg magnesium sulfate heptahydrate
  • 100mg calcium chloride dihydrate

Use deionized or distilled water; occasionally problems arise with less pure sources.

M13 Sweet

This is our production medium.

After autoclaving a liter of de-ionized water in a flask, add

  • 40mL 25X M13 concentrate
  • 10mL Hutner's Mineral Base Concentrate

We sometimes add a little extra calcium chloride (100mM stock to a final concentration of 1mM) to the flasks to encourage aggregates.

When the 1X M13 Sweet medium is assembled as indicated above, the final concentrations are:

  • 0.5% glucose
  • 0.15% glutamate
  • 2mM phosphate
  • 5 mM imidazole
  • 0.04% ammonium chloride
  • 1% Hutner's Mineral Salts

25X M13 concentrate

Component stock solutions:

  • 125.0g glucose (or 137.5g glucose monohydrate) in a total volume of 600mL dH2O
  • 47.7g L-glutamic acid sodium salt (MW 187.13) in a total volume of 200mL dH2O
  • 8.5g imidazole in a total volume of 100mL dH2O
  • 3.4g KH 2PO4 in a volume of 50mL dH2O
  • 10g ammonium chloride in a total volume of 50mL dH2O

All component stocks should be made in separate bottles, autoclaved, and once they are cooled, combined into a single sterile bottle. If the components are mixed first and then autoclaved, the stock turns black. Store the stock at room temperature or at 4C. Don't worry if after a few weeks the 25X stock turns color from clear-yellow to yellow-brown.

SOB Medium

Used in growing bacteria for preparing chemically competent cells

Ingredients

  • 0.5% (w/v) yeast extract
  • 2% (w/v) tryptone
  • 10 mM NaCl
  • 2.5 mM KCl
  • 20 mM MgSO4

Per liter:

  • 5 g yeast extract
  • 20 g tryptone
  • 0.584 g NaCl
  • 0.186 g KCl
  • 2.4 g MgSO4
  • Note: Some formulations of SOB use 10 mM MgCl2 and 10 mM MgSO4 instead of 20 mM MgSO4.
  • SOB medium is also available dry premixed from Difco. Adjust to pH 7 prior to use. This requires approximately 25 ml of 1M NaOH per liter.

SOC Medium(per 100 ml)

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ote This medium should be prepared immediately before use.

  • Add 2 ml of filter-sterilized 20% (w/v) glucose or 1 ml of filter-sterilized 2 M glucose to 100 ml sterile SOB
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