IGEM:PennState/2006/Growthmedia: Difference between revisions

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<h1>Growth Media and plates</h1>
<h1>Growth Media</h1>
 
<h2>LB</h2>
===<font color="red">LB Media</font>===
<h3>Media (per liter)</h3>
(Per L)
*10 g Bactotryptone
*10 g bactotryptone
*5 g Yeast Extract
*5 g yeast extract
*10 g NaCl
*10 g NaCl
*pH to 7.0 (~40uL 2M NaOH / L)
*pH to 7.0 with NaOH/HCl
*Autoclave
<h4>Plates</h4>
*Add 15 g Bacto-Agar per liter media
*Autoclave for 25 minutes
**If antibiotics necessary, add only once media has cooled to ~45ºC, or is comfortable to the touch
*Pour in sterile environment (~15ml per plate)
*Cool in such a way as to avoid accumulation of condensation, while maintaining sterility
*Seal with parafilm, place in bag, and store at 4ºC


===<font color="red">M9 Minimal Media</font>===
<h2>LBM</h2>
(SUPPLEMENTED W/ 0.1% YE)
<h3>Media (per liter)</h3>
Final concentrations:
*10 g Bactotryptone
*1X M9 salts (autoclaved)
*5 g Yeast Extract
*1 mM MgSO4 (autoclaved)
*5 g NaCl
*0.16% glycerol (autoclaved)
*0.4066 g MgCl<sub>2</sub>.6H<sub>2</sub>O (or 2mL 1M MgCl<sub>2</sub>)
*0.148 μM thiamine/vitamin B1 (filter sterilized)
*pH to 7.0 with NaOH/HCl
*0.1% casamino acids (filter sterilized)
*Autoclave
*0.1% Yeast Extract (autoclaved)
<h4>Plates</h4>
===<font color="green">Procedure:</font>===
*Add 15 g Bacto-Agar per liter media
1. These solutions should already be made,
*Autoclave for 25 minutes
so add together w/sterile technique:
**If antibiotics necessary, add only once media has cooled to ~45ºC, or is comfortable to the touch
*Pour in sterile environment (~15ml per plate)
*Cool in such a way as to avoid accumulation of condensation, while maintaining sterility
*Seal with parafilm, place in bag, and store at 4ºC
<h4>Top Agar (per liter)</h4>
*500 mL LBM
*500 mL DI H<sub>2</sub>O
*8 g Bacto-Agar
*Autoclave


*200 mL 5X M9 salts
<h2>SOB</h2>
*1 mL 1 M MgSO4
<h3>Media (per liter)</h3>
*2.27 mL 70% glycerol
*20 g bactotryptone
*50 μL thiamine (10mg/mL)
*5 g bacto-yeast extract
*5 mL casamino acids (200 mg/mL)
*0.5 g NaCl
*10 mL 0.1g/mL Yeast Extract
*0.19 g KCl
*782 mL sterile dH2O                          .
*pH to 7.0 with NaOH/HCl
*1 L M9 media (for plates just add 15 g bacto agar to recipe)
*Autoclave for 25 minutes
 
*Add filter-sterilized MgCl<sub>2</sub> and MgSO<sub>4</sub> to yield final concentration of 10 mM each
===<font color="red">SOB Media</font>===
<h4>Final Concentration:</h4>
Final concentrations
*0.5% Yeast Extract
*0.5% Yeast Extract
*2% Tryptone
*2% Tryptone
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*10 mM MgSO4
*10 mM MgSO4


===<font color="green">Procedure</font>===
<h2>SOC</h2>
1. To 1 L, add:
<h3>Media</h3>
*20 g bactotryptone
*Add filter-sterilized glucose to SOB for 20 mM final concentration of glucose
*5 g bacto-yeast extract
*0.5 g NaCl
*0.19 g KCl
*Adjust to pH 7.0 w/ NaOH
 
2. Autoclave on slow exhaust for 25 min.
 
3. Add filter-sterilized MgCl2, MgSO4 solution to give final Mg2+ conc. of 20 mM (i.e. 10 mM MgCl2, 10 mM MgSO4)
 
SOC MEDIA
SOB + 20 mM (final) glucose (aka dextrose)
''
 
='''LB Plates'''=
 
===<font color="red">LB Plates</font>===
(per/L H2O)
1. Weigh and add to H2O in large Erlenmeyer:
*10 g Bacto-tryptone
*5 g Bacto-yeast extract
*10 g NaCl
*15 g Bacto-agar
 
2. Autoclave on slow exhaust cycle 25 min
 
3. Cool in tap-water-bath 10 min, then place in 45ºC water bath.
(in sterile hood)
 
4. When media has cooled to ~45ºC (comfortable to hold), add appropriate antibiotic. 
 
5. Pour plates (~25 mL/plate)
 
6. Allow to solidify & store in bag in cold-room


[http://openwetware.org/wiki/IGEM:PennState Main]
<h2>Minimal Media</h2>
<h3>M9 Salts (per liter)</h3>
*  1. Make M9 salts
*  2. 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
<h4>Media (per liter)</h4>
*  3. Measure ~700ml of distilled H2O (sterile)
*  4. Add 200ml of M9 salts
*  5. Add 2ml of 1M MgSO4 (sterile)
*  6. Add 20 ml of 20% glucose (or other carbon source)
*  7. Add 100ul of 1M CaCl2 (sterile)
*  8. Adjust to 1000ml with distilled H2O

Latest revision as of 06:39, 1 July 2008

Growth Media

LB

Media (per liter)

  • 10 g Bactotryptone
  • 5 g Yeast Extract
  • 10 g NaCl
  • pH to 7.0 with NaOH/HCl
  • Autoclave

Plates

  • Add 15 g Bacto-Agar per liter media
  • Autoclave for 25 minutes
    • If antibiotics necessary, add only once media has cooled to ~45ºC, or is comfortable to the touch
  • Pour in sterile environment (~15ml per plate)
  • Cool in such a way as to avoid accumulation of condensation, while maintaining sterility
  • Seal with parafilm, place in bag, and store at 4ºC

LBM

Media (per liter)

  • 10 g Bactotryptone
  • 5 g Yeast Extract
  • 5 g NaCl
  • 0.4066 g MgCl2.6H2O (or 2mL 1M MgCl2)
  • pH to 7.0 with NaOH/HCl
  • Autoclave

Plates

  • Add 15 g Bacto-Agar per liter media
  • Autoclave for 25 minutes
    • If antibiotics necessary, add only once media has cooled to ~45ºC, or is comfortable to the touch
  • Pour in sterile environment (~15ml per plate)
  • Cool in such a way as to avoid accumulation of condensation, while maintaining sterility
  • Seal with parafilm, place in bag, and store at 4ºC

Top Agar (per liter)

  • 500 mL LBM
  • 500 mL DI H2O
  • 8 g Bacto-Agar
  • Autoclave

SOB

Media (per liter)

  • 20 g bactotryptone
  • 5 g bacto-yeast extract
  • 0.5 g NaCl
  • 0.19 g KCl
  • pH to 7.0 with NaOH/HCl
  • Autoclave for 25 minutes
  • Add filter-sterilized MgCl2 and MgSO4 to yield final concentration of 10 mM each

Final Concentration:

  • 0.5% Yeast Extract
  • 2% Tryptone
  • 10mM NaCl
  • 2.5 mM KCl
  • 10 mM MgCl2
  • 10 mM MgSO4

SOC

Media

  • Add filter-sterilized glucose to SOB for 20 mM final concentration of glucose

Minimal Media

M9 Salts (per liter)

  • 1. Make M9 salts
  • 2. 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

Media (per liter)

  • 3. Measure ~700ml of distilled H2O (sterile)
  • 4. Add 200ml of M9 salts
  • 5. Add 2ml of 1M MgSO4 (sterile)
  • 6. Add 20 ml of 20% glucose (or other carbon source)
  • 7. Add 100ul of 1M CaCl2 (sterile)
  • 8. Adjust to 1000ml with distilled H2O
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