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General Zamanian Lab Recipes

Ampicillin Stock (100 mg/mL)

  1. Mix 2 g of ampicillin powder (Fisher AC611770250) into 20 mL of MilliQ H2O
  2. Vortex until the ampicillin is dissolved
  3. Sterilize using a 50 mL syringe filter into a 50 mL conical tube

  4. Aliquot 1 mL of the ampicillin solution into labeled 1.5 mL tubes and store at -20°C

  5. Note: Filtering and aliquoting should be performed using sterile techniques in the hood

Kanamycin Stock (50 mg/mL)

  1. Mix 500 mg of kanamycin powder (Fisher AC450810100) into 9 mL of MilliQ H2O
  2. Vortex until kanamycin is dissolved
  3. Aliquot 1 mL of the kanamycin solution into labeled 1.5 mL tubes and store at -20°C

LB Miller Broth

  1. Mix the following:

    Reagent Amount
    Tryptone 10 g
    Yeast extract 5 g
    NaCl 10 g
    dH2O up to 1 L

  2. Autoclave on liquid cycle for 30 min

  3. Add antibiotics after media has cooled (if needed):

    Antibiotic Stock Dilution Final Volume (for 500 mL media)
    Ampicillin 100 mg/mL 1:1000 100 μg/mL 0.5 mL
    Kanamycin 50 mg/mL 1:1000 50 μg/mL 0.5 mL
    Chloramphenicol 50 mg/mL 1:3333 15 μg/mL 0.15 mL

  4. Store media with antibiotics at 4°C

LB Miller Agar

  1. Mix the following:

    Reagent Amount
    Tryptone 10 g
    Yeast extract 5 g
    Bacto Agar 15 g
    NaCl 10 g
    dH2O up to 1 L

  2. Autoclave on liquid cycle for 30 min

  3. Place media in a 55°C waterbath for 1 hr

  4. Once cooled, pour media or add antibiotics

    Antibiotic Stock Final Volume (for 500 mL media)
    Ampicillin 100 mg/mL 100 μg/mL 0.5 mL
    Kanamycin 50 mg/mL 50 μg/mL 0.5 mL
    Chloramphenicol 50 mg/mL 15 μg/mL 0.15 mL

  5. Mix well after adding antiobiotic and then pour

  6. Optional: If needed for blue/white screening, add X-Gal and IPTG:

    Antibiotic Stock Final Volume (for 500 mL media)
    X-Gal (reconstituted in DMSO) 50 mg/mL 200 μg/mL 2 mL
    IPTG (reconstituted in Water) 100 mM 1 mM 5 mL

  7. Store plates at 4°C

50X TAE

  1. Mix the following:

    Reagent Amount
    Tris Base 242 g
    EDTA (0.5 M pH 8) 100 mL
    Glacial Acetic Acid 57.1 mL
    dH2O up to 1 L

  2. Be very careful when pipetting the glacial acetic acid, as it is very caustic.

  3. Use a GLASS pipette

EDTA 0.5 M (for 50X TAE, TE Buffer)

Reagent Amount
EDTA, disodium salt 93 g
NaOH pellets ~ 10 g
dH2O up to 500 mL
  1. Add EDTA powder to ~450 mL of dH2O while stirring and monitoring pH
  2. Slowly add the NaOH pellets until pH = 8
  3. Bring the volume up to 500 mL
  4. Filter sterilize

TRIS 1M pH 8 (for 50x TAE, TE Buffer)

Reagent Amount
Tris Base 121.14 g
HCl ~ 80-85 mL
dH2O up to 1 L
  1. Dissolve Tris Base in 800 mL dH2O, while monitoring pH.
  2. Slowly add HCl until pH = 8
  3. Bring final volume to 1 L
  4. Aliquot 200 mL into 250 mL bottles
  5. Autoclave on liquid cycle for 30 minutes

TE Buffer

  1. Mix the following:

    Reagent Amount (500 mL total) Amount (200 mL total)
    1M Tris (pH 8) 5 mL 2 mL
    0.5M EDTA (pH 8) 5 mL 2 mL
    dH2O 494 mL 197.6 mL

  2. Aliquot 10 mL each into 15 mL conical tubes

Orange G 6X Gel Loading Dye

  1. Make a 6X stock (0.9%) by adding Orange G dye to glycerol in a 15 mL conical tube and mixing completely:

    Reagent Amount
    Orange G dye 45 mg
    30% Glycerol (filter sterilized) 10.0 mL

  2. Aliquot 1 mL each into pre-labeled 1.7 mL tubes and store at room temperature in labeled box. Additional tubes can be stored at -20°C.

TSS Buffer (for chemically competent bacteria)

  1. Mix the following:

    Reagent Amount
    PEG 8000 (or 3350) 5 g
    MgCl2 * 6H2O 0.3 g
    DMSO 2.5 mL
    LB Broth up to 50 mL

  2. Filter sterilize through 0.22 um filter

SOC (for bacterial transformations)

  1. Mix the following:

    Reagent 1 L Stock 500 mL Stock 250 mL Stock
    Yeast extract 5 g 2.5 g 1.25 g
    Tryptone 20 g 10 g 5 g
    NaCl 0.584 g 0.292 g 0.146
    KCl 0.186 g 0.093 g 0.0465 g
    MgSO4 2.4 g 1.2 g 0.6 g
    dH2O up to 1 L up to 500 mL up to 250 mL

  2. Autoclave on liquid cycle for 30 min

  3. Adjust pH to 7.5 prior to use (this requires ~ 25 mL of 1M NaOH per liter)

  4. Add filter-sterilized (0.22 um filter) 20% Glucose solution (20 g in 100 mL dH2O) after the media has cooled:

    Stock Vol. 20% Glucose Amount
    1 L 20 mL
    500 mL 10 mL
    250 mL 5 mL

  5. Store at room temperature or at +4°C.

1P2P (1.4% stock solution)

  1. Mix the following:
Reagent Amount
93% 1P2P stock 15 ml
M9 985 ml
  1. Be sure to mix well when making stock and before each use
  2. Store at room temperature