Team:Brown-Stanford/Lab/Protocols/LB

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{{:Team:Brown-Stanford/Templates/Main}}
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{{:Team:Brown-Stanford/Templates/Protocol}}
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<html><h1>Protocol Code: M2</h1></html>
== '''LB (Lysogeny broth)''' ==
== '''LB (Lysogeny broth)''' ==
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=== Making the broth ===
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#Add 250 mL of dH2O to a graduated cyclindar.
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''(Taken from <html><a href=""http://www.brown.edu/Research/Wessel_Lab/"">Wessel Lab at Brown University</a></html>)''
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The following protocol makes ~40 plates.
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=== '''Making the broth''' ===
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#Add 250 mL of dH<sub>2</sub>O to a graduated cyclindar.
#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)}}
#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)}}
#Mix powder well to bring into solution
#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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#Add dH<sub>2</sub>O to total volume of 500 mL and transfer to 1 L flask
#Put on stirring hot plate and heat to boil for 1 min while stirring.
#Put on stirring hot plate and heat to boil for 1 min while stirring.
#Transfer to 1 L pyrex jar and label with autoclave tape.
#Transfer to 1 L pyrex jar and label with autoclave tape.
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#Let liquid cool to ~55C (you should be able to pick up the jar without a glove)
#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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=== '''Pouring plates''' ===
#Make sure bench top has wiped down with bleach/EtOH.
#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
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#Remove sterile Petri dishes (VWR 25384-208) from plastic bag (save the bag for storage).
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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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#Pour a thin layer (5mm) of LB Agar (~10mL) into each plate being careful to not
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-
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.
#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
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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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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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#Store plates in plastic bags in fridge with: name, date and contents (note any
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additive).
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== Special Additives ==
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=== '''Special Additives''' ===
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''to be added to LB Agar right before pouring plates''
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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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*Ampicillin (VWR 80055-786) 50 mg dissolved in a small amout of dH<sub>2</sub>O (concentration 100 ug/mL)
*X-gal (VWR IB02260) 50 mg dissolved in a small amouth of DMSO
*X-gal (VWR IB02260) 50 mg dissolved in a small amouth of DMSO
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== Stock solutions ==
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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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*Ampicillin 20mg/mL 200mg in 10mL dH<sub>2</sub>O (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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*IPTG (VWR EM-5800) 100mM 238 mg IPTG in 10mL dH<sub>2</sub>O (store at –20 in 1mL aliquots) use 40uL on each plate
*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
*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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{{:Team:Brown-Stanford/Templates/Foot}}
{{:Team:Brown-Stanford/Templates/Foot}}

Latest revision as of 18:16, 28 September 2011

Brown-Stanford
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Protocol Code: M2

LB (Lysogeny broth)

(Taken from Wessel Lab at Brown University)

The following protocol makes ~40 plates.

Making the broth

  1. Add 250 mL of dH2O to a graduated cyclindar.
  2. Mix one of the following:
    1. Weigh out 20g of premix LB Agar powder (VWR DF0445-17)
    1. 5.0 g tryptone
    2. 2.5 g yeast extract
    3. 5.0 g NaCl
    4. 7.5 g agar (if making agar plates)
  1. Mix powder well to bring into solution
  2. Add dH2O to total volume of 500 mL and transfer to 1 L flask
  3. Put on stirring hot plate and heat to boil for 1 min while stirring.
  4. Transfer to 1 L pyrex jar and label with autoclave tape.
  5. Autoclave at liquid setting for 20 minutes in a basin making sure to loosen top
  6. Let liquid cool to ~55C (you should be able to pick up the jar without a glove)

Pouring plates

  1. Make sure bench top has wiped down with bleach/EtOH.
  2. Remove sterile Petri dishes (VWR 25384-208) from plastic bag (save the bag for storage).
  3. 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).
  4. Swirl plate in a circular motion to distribute agar on bottom completely.
  5. Let each plate cool until its solid (~20 minutes) then flip so as to avoid condensation on the agar.
  6. Store plates in plastic bags in fridge with: name, date and contents (note any additive).

Special Additives

(To be added to LB Agar right before pouring plates)

  • Ampicillin (VWR 80055-786) 50 mg dissolved in a small amout of dH2O (concentration 100 ug/mL)
  • X-gal (VWR IB02260) 50 mg dissolved in a small amouth of DMSO

Stock solutions

  • Ampicillin 20mg/mL 200mg in 10mL dH2O (store at 4 in 1mL aliquots) use 50uL on each plate
  • IPTG (VWR EM-5800) 100mM 238 mg IPTG in 10mL dH2O (store at –20 in 1mL aliquots) use 40uL on each plate
  • 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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