Team:Brown-Stanford/Lab/Protocols/ATCC1376

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<html><h1>Protocol Code: M3</h1></html>
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== '''ATCC medium: 1376 ''Bacillus pasteurii'' NH4-YE medium''' ==
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== '''ATCC medium: 1376 (''Bacillus pasteurii'' NH<sub>4</sub>-YE medium)''' ==
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=== '''Making the broth''' ===
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#Add 250 mL of dH2O to a graduated cyclindar.
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#Mix one of the following:{{Multi-column numbered list|lst=disc||Weigh out 20g of premix LB Agar powder (VWR DF0445-17)||<li>5.0 g tryptone<li>2.5 g yeast extract<li>5.0 g NaCl<li>7.5 g agar (if making agar plates)}}
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#Mix powder well to bring into solution
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#Add dH2O to total volume of 500 mL and transfer to 1 L flask
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#Put on stirring hot plate and heat to boil for 1 min while stirring.
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#Transfer to 1 L pyrex jar and label with autoclave tape.
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#Autoclave at liquid setting for 20 minutes in a basin making sure to loosen top
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#Let liquid cool to ~55C (you should be able to pick up the jar without a glove)
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=== '''Pouring plates''' ===
 
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#Make sure bench top has wiped down with bleach/EtOH.
 
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#Remove sterile Petri dishes (VWR 25384-208) from plastic bag (save the bag for storage).
 
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#Pour a thin layer (5mm) of LB Agar (~10mL) into each plate being careful to not lift the cover off excessively (you should be able to just open up enough to pour).
 
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#Swirl plate in a circular motion to distribute agar on bottom completely.
 
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#Let each plate cool until its solid (~20 minutes) then flip so as to avoid condensation on the agar.
 
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#Store plates in plastic bags in fridge with: name, date and contents (note any additive).
 
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=== '''Special Additives''' ===
 
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(''To be added to LB Agar right before pouring plates'')
 
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*Ampicillin (VWR 80055-786) 50 mg dissolved in a small amout of dH2O (concentration 100 ug/mL)
 
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*X-gal (VWR IB02260) 50 mg dissolved in a small amouth of DMSO
 
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=== '''Stock solutions''' ===
 
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*Ampicillin 20mg/mL 200mg in 10mL dH2O (store at 4 in 1mL aliquots) use 50uL on each plate
 
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*IPTG (VWR EM-5800) 100mM 238 mg IPTG in 10mL dH2O (store at –20 in 1mL aliquots) use 40uL on each plate
 
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*X-gal 40 mg/mL 400 mg X-gal in 10mL DMSO (store at –20 in 1mL aliquots foil wrapped tubes) use 40uL on each plate
 
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*20.0 g Yeast extract
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*10.0 g (75.7 mM) (NH<sub>4</sub>)2SO<sub>4</sub>
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*1.0 L 0.13 M Tris buffer (pH 9.0)
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*20.0 g Agar (if needed)
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Autoclave ingredients separately. No growth occurs when ingredients are sterilized together.
{{:Team:Brown-Stanford/Templates/Foot}}
{{:Team:Brown-Stanford/Templates/Foot}}

Latest revision as of 18:17, 28 September 2011

Brown-Stanford
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Protocol Code: M3

ATCC medium: 1376 (Bacillus pasteurii NH4-YE medium)

  • 20.0 g Yeast extract
  • 10.0 g (75.7 mM) (NH4)2SO4
  • 1.0 L 0.13 M Tris buffer (pH 9.0)
  • 20.0 g Agar (if needed)

Autoclave ingredients separately. No growth occurs when ingredients are sterilized together.

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