Compiled By: Laurie Brier

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Stock Reagent Recipes

• Compiled By Laurie Brier

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Table of contents
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Table of Contents (Cont.)
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Table of Contents (Cont.)
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S Basal Medium

• Start with a bottle of 500 mL of distilled water.
• Add
• 2.93 g of NaCl
• 0.5 g of K2HPO4
• 3 g of KH2PO4
• 500 µL of Cholesterol
• Autoclave the solution.
• Makes 500 mL.

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1 M Anhydrous CaCl2

• Dissolve 11.099 g of CaCl2 into 100 mL of
  distilled water.
• Makes 100 mL

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Cholesterol

• Autoclave 250 mL bottles.
• Use a graduated cylinder to pour 50 mL of 95
  ETOH into a sterile falcon tube. Also, measure
  0.25 g of cholesterol into a clean weigh boat.

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Cholesterol (Cont.)

• Pour the ETOH into a 250 ml bottle and add the
  cholesterol.
• Close the bottle and shake to mix.
• Make sure the solution is clear or heat the
  solution to make sure the cholesterol is in
  solution.
• Makes 50 mL.

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0.5 Gelatin

• Place 0.5 g of dry, solid gelatin 100 mL of
  distilled water.
• Autoclave the mixture.
• Makes 100 mL.

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30 Glycerin

• Mix 30 mL of glycerin with 70 mL of distilled
  water.
• Autoclave the solution.
• Makes 100 mL

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500 mM KCl

• Dissolve 3.728 g of KCl into 100 mL of distilled
  water.
• Autoclave the solution.
• Makes 100 mL.

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1 M KH2PO4 (pH 6)

• Dissolve 17.418g KH2PO4 into 100 mL of distilled
  water.
• Use 5 M NaOH to bring the pH to 6.
• Makes 100 mL

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25 mM MgCl

• Dissolve 0.508 g of MgCl into 100 mL of distilled
  water.
• Autoclave the solution.
• Makes 100 mL.

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1 M MgSO4

• Dissolve 12.037g MgSO4 into 100mL of water.
• Makes 100 mL

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1 M NaCl

• Dissolve 5.844 g of NaCl into 100 mL of water.
• Makes 100 mL

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NaOH Solutions

• Use 100 mL of distilled water.
• For 1 N NaOH, add 4 g of NaOH.
• For 10 N NaOH, add 40 g of NaOH.
• Use caution. The solution could get very hot!
• Makes 100 mL.

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PCR Lysis Buffer

• Into a small, sterile tube mix
• Pro K (at 1 mg/ml) ? 3 µL
• 100 mM Tris ? 100 µL
• 500 mM KCl ? 100 µL
• 25 mM MgCl2 ?100 µL
• 4.5 Tween ? 100 µL
• 0.5 Gelatin ? 100 µL
• Distilled water ? 497 µL
• Makes 1 mL

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Pro K

• Mix 2 µL of stock Pro K (20 mg/ml) with 38 µL of
  distilled water.
• The Pro K is now at a concentration of 1 mg/ml
• Aliquot this new diluted Pro K solution into
  sterile microtubes.

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1 X TBE

• Dilute 100 mL of 10 X TBE with 900 mL of
  distilled water
• Makes 1 Liter

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100 mM Tris

• Dissolve 1.214 g of Tris into 100 mL of distilled
  water.
• Autoclave the solution.
• Makes 100 mL.

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4.5 Tween

• Dissolve 4.5 mL of Tween 20 into 95.5 mL of
  distilled water.
• Autoclave the solution
• Makes 100 mL.

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2X YTB

• Into an empty bottle add
• 4 g of tryptone
• 2.5 g of yeast extract
• 1.25 g of NaCl
• Then add 250 mL of distilled water.
• Autoclave the solution.
• Place 10 mL of the autoclaved solution into each
  of 25 sterile test tubes.

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Inoculating OP-50 bacteria into 2X YTB

• Place 10 ml of 2X YTB into a sterile 15 ml tube
• Fetch a microtube of OP-50 bacteria from the
  freezer.
• Using the aseptic technique, inoculate the 2X YTB
  buffer with a loopful of bacteria.
• Leave the tubes in an incubator (37 C) overnight.