Team:Brown-Stanford/Lab/Protocols/LB

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== '''LB (Lysogeny broth)''' ==
== '''LB (Lysogeny broth)''' ==
 +
''(Taken from [http://www.brown.edu/Research/Wessel_Lab/ Wessel Lab at Brown University])''
 +
=== '''Making the broth''' ===
=== '''Making the broth''' ===
#Add 250 mL of dH2O to a graduated cyclindar.
#Add 250 mL of dH2O to a graduated cyclindar.

Revision as of 23:44, 11 June 2011

Brown-Stanford
iGEM

LB (Lysogeny broth)

(Taken from [http://www.brown.edu/Research/Wessel_Lab/ Wessel Lab at Brown University])

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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