Griffitts:Stock solutions

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Contents

Antibiotics

Ampicillin (40 mL)

  • 1 g Ampicillin
  • 40 mL ddH2O
  • Filter sterilize
    • Store in refrigerator

Chloramphenicol (40 mL)

Erythromycin (20 mL)

  • 1.0 g Erythromycin
  • 20 mL 100% ethanol (in fume hood)
  • Do not filter sterilize—the ethanol may dissolve the filter
    • Store in the -20°C Freezer

Gentamycin (40 mL)

  • 0.3 g Gentamycin
  • 40 mL ddH2O
  • Filter sterilize
    • Store in refrigerator

Hygromycin (40 mL)

Kanamycin (40 mL)

  • 0.1 g Kanamycin
  • 40 mL ddH2O
  • Filter sterilize
    • Store in refrigerator

Nalidixic acid (30 mL)

  • 0.9 g Nalidixic Acid
  • 0.36 g NaOH
  • 30 mL ddH2O
  • Filter sterilize
    • Store in refrigerator

Neomycin (40 mL)

  • 0.8 g Neomycin
  • 40 mL ddH2O
  • Filter sterilize
    • Store in refrigerator

Rifampicin (20 mL)

  • 1.0 g Rifampicin
  • 20 mL N,N-dimethylformamide (DMF; in fume hood)
  • Do not filter sterilize—the DMF will dissolve the filter
    • Store in the -20°C Freezer

Spectinomycin (40 mL)

  • 0.8 g Spectinomycin
  • 40 mL ddH2O
  • Filter sterilize
    • Store in refrigerator

Streptomycin (40 mL)

  • 2.0 g Streptomycin
  • 40 mL ddH2O
  • Filter sterilize
    • Store in refrigerator

Tetracycline (40 mL)




Antibiotic Specifications
Antibiotic Abbr. Stripe Stock E. coli S. meliloti
Ampicillin Ap BLACK 25 mg/mL 100 μg/mL (250X) N/A
Chloramphenicol Cm BROWN 7.5 mg/mL 30 μg/mL (250X) N/A
Gentamycin Gm PURPLE 7.5 mg/mL 3 μg/mL (2500X) 15 μg/mL (500X)
Hygromycin Hy (none) 5 mg/mL 20 μg/mL (250X) 20 μg/mL (250X)
Kanamycin Km YELLOW 7.5 mg/mL 30 μg/mL (250X) N/A
Neomycin Nm GREEN 20 mg/mL N/A 80 μg/mL (250X)
Spectinomycin Sp ORANGE 20 mg/mL 80 μg/mL (250X) 80 μg/mL (250X)
Streptomycin Sm BLUE 50 mg/mL N/A 200 μg/mL (250X)
Tetracycline Tc RED 2.5 mg/mL 10 μg/mL (250X) 10 μg/mL (250X)

Note: These parameters are intended to be compatible with pre-made 250-mL amounts of LB agar, such that usually 1 mL of stock solution is diluted into the LB agar. For liquid LB (typically 75 mL amounts) 0.3 mL of the stock solution will usually be added. Exception: Gentamycin for E. coli.

DNA

10 mM dNTPs

  • Thaw and mix (stir) 100 mM stock solutions of G, A, T, C (NEB)
  • Combine in a 1.5-mL tube:
    • 60 μl H2O
    • 10 μl G
    • 10 μl A
    • 10 μl T
    • 10 μl C

Note: Store in the -20°C freezer in the Box marked "Other Enzymes"

10X Taq buffer

Note: Store in the -20°C freezer in the Box marked "Other Enzymes"

10X Pfx buffer

Note: Store in the -20°C freezer in the Box marked "Other Enzymes"

Media Additives

1 M Calcium Chloride (75 mL)

  • 75 mL ddH2O
  • 11.027 g CaCl2·2H2O

Autoclave

2% X-gal

  • 10 mL N,N-dimethylformamide (DMF)(in fume hood)
  • 0.2 g 5-bromo-4-chloro-3-indolyl- beta-D-galactopyranoside (X-gal)(in freezer)

Store at -20°C

0.2 M IPTG

  • 5 mL ddH2O
  • 0.238g isopropyl-β-D-thiogalactopyranoside (IPTG)(in freezer)

Sterile filter, aliquot, and store at -20°C

Copper sulfate

20% SDS (75 mL)

  • 75 mL ddH2O
  • 15 g Sodium dodecyl sulfate (SDS)

Do not sterilize

30% Acrylamide (75 mL)

  • 75 mL ddH2O
  • 22.5 g acrylamide

10% Ammonium persulfate (75 mL)

  • 75 mL ddH2O
  • 7.5 g ammonium persulfate (APS)

5 M NaCl (75 mL)

  • 75 mL ddH2O
  • 21.915 g NaCl

Autoclave

80% Glycerol (75 mL)

  • 75 mL ddH2O
  • 60 g glycerol

Note: use heating to mix
Autoclave

Orcinol spray

  • 5 mL ddH2O
  • 25 mL methanol (in fume hood)
  • 10 mL sulfuric acid (H2SO4 (in fume hood—be careful!)
  • 200 mg orcinol
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