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Zamanian Lab C. elegans Recipes

M9 Buffer (4x Stock)

  1. Mix the following to make 4x stock solutions:

    Reagent Amount for 4L
    KH2PO4 48 g
    Na2HPO4 96 g
    NaCl 80 g
    MQ H2O up to 4 L

  2. To make 4 L of 4x stock:

    a. Measure all reagents into 3 L water in a 6 L flask and fully dissolve. Then split the volume into two 2-liter graduated cylinders and bring volume of each to 2 L. Combine in 6 L flask and aliquot into 8 500 mL aliquots.

  3. Autoclave on liquid cycle for 1 hr. Make sure to loosen the cap only a little to maintain final volume.

  4. When the M9 is cool, add: 2 mL sterile MgSO4 1 M per 500 mL of M9 buffer.

Note: use autoclaved 2 L water bottles (1500 mLs) to dilute to 1x for use in assays.

MgSO4, 1 M Solution (for M9, S Medium)

  1. To make 500 mL, completely dissolve 123.24 g of MgSO4 heptahydrate (FW= 246.48) in 450 mL distilled water (from the carboy). Transfer to a graduated cylinder and bring up to the final volume with distilled water.

  2. Autoclave for 1 hr.

Potassium Phosphate Buffer, 1 M pH 6 (for S Basal/Medium, Freezing Solution)

  1. Mix the following:

    Reagent Amount Needed
    KH2PO4 136.1 g
    KOH 17.99 g
    MQ H2O up to 1 L

  2. Autoclave for 1 hr. Store in 500 mL aliquots.

S Basal

  1. Mix the following:

    Reagent Amount Needed
    NaCl, 5 M 20 mL
    Potassium Phosphate Buffer, 1 M pH 6 50 mL
    Cholesterol, 5 mg/mL in ethanol 1 mL
    MQ H2O 930 mL

  2. Autoclave on liquid cycle for 1 hr. (Note that the solution will be cloudy)

K Medium

  1. Mix the following pre-autoclaved salts:

    Reagent Amount Needed
    NaCl, 5 M 5.1 mL
    KCl, 1 M 16 mL
    CaCl2, 1 M 1.5 mL
    MgSO4, 1 M 1.5 mL
    MQ H2O 500 mL

  2. Filter sterilize using a 0.22 μm filter.

  3. Add 125 μL of 5mg/mL Cholesterol after filtering.

  4. Store at RT for up to 2 weeks.

S Medium

  1. Make each component below according to the recipe.

  2. Using sterile technique, mix the following:

    Reagent Amount Needed
    NaCl, 5 M 40 mL
    Potassium Phosphate Buffer, 1 M pH 6 20 mL
    Trace Metals Solution 20 mL
    CaCl2, 1 M 6 mL
    MgSO4, 1 M 6 mL
    Phosphate Buffer, 1 M 20 mL
    MQ H2O 1886 mL

  3. Filter sterilize using a 0.22 μm filter in 499.5 mL aliquots.

  4. Using good sterile technique, add 0.5 mL of cholesterol (5 mg/mL in EtOH) to each aliquot.

Trace Metals Solution (for S Medium)

  1. Dissolve 3.06 g Trace Metals Mix (US Biological, cat # N1010) into 1 L of MQ H2O.

  2. Stir with heat until completely solubilized.

  3. Aliquot 100 mL into amber PDPE bottles.

  4. Autoclave on liquid cycle for 1 hr., using the large autoclave on the left.

  5. Be careful to just place the cap on top of the bottle

  6. If you screw the cap on, the bottle could deform during autoclaving.

Potassium Citrate, 1 M pH 6 (for S Medium)

  1. Mix the following:

    Reagent Amount Needed
    Citric Acid Monohydrate 20 g
    Tri-potassium Citrate Monohydrate 293.5 g
    MQ H2O up to 1 L

  2. Autoclave on liquid cycle for 1 hr.

CaCl2, 1 M Solution (for S Medium)

  1. To make 500 mL, completely dissolve 73.5 g of CaCl2 dehydrate (FW=147.01) in 450 mL water (from the carboy). Transfer to a graduated cylinder and bring up to final volume.

  2. Autoclave for 1 hr.

NaCl, 5 M Solution

  1. To make 1 L, dissolve 292 g of NaCl (FW=58.44) in 900 mL distilled water (from the carboy). Transfer to a graduated cylinder and slowly bring up to final volume to 1L to achieve complete dissolution. This is stored without sterilizing.

KCl, 1M Solution

  1. To make 500 mL, completely dissolve 37.28 g of KCl (FW=74.55) in 450 mL distilled Milli-Q water. Transfer to a graduated cylinder and bring up to final volume.

  2. Autoclave for 1 hr.

Freezing Solution

  1. Mix the following:

  2. Dissolve the glycerol in water first, then mix in other reagents.

    Reagent Amount Needed
    Potassium phosphate buffer, 1 M pH 6 100 mL
    NaCl, 5 M 40 mL
    Glycerol, 100% 600 mL
    MQ H2O up to 2 L

  3. Divide into 200 mL aliquots in 250 mL glass square bottles.

  4. Autoclave on liquid cycle for 1 hr.

  5. IMMEDIATELY BEFORE USE: Add MgSO4 to a final concentration of 0.3 mM (e.g. for 200 mL of freezing solution, add 60 μL of 1 M MgSO4).

LB Miller Broth

  1. Mix the following:

    Reagent Amount Needed
    Tryptone 10 g
    Yeast Extract 5 g
    NaCl 10 g
    MQ H2O up to 1 L

  2. Autoclave on liquid cycle for 1 hr.

  3. Once the media is cooled after autoclaving, you can add antibiotics (if needed):

    Antibiotic Stock Dilution Final Concentration Volume Added to 500 mL of 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
    Chloramphenical 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 Needed
    Tryptone 10 g
    Yeast Extract 5 g
    NaCl 10 g
    Agar 15 g
    MQ H2O up to 1 L

  2. Autoclave on liquid cycle for 1 hr.

  3. Once the media is out of the autoclave, place it in a 55°C water bath for 1 hr.

  4. After the media is cooled down to 55°C, you can either pour right away, or add the antibiotic of your choice:

    Antibiotic Stock Dilution Final Concentration Volume Added to 500 mL of 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
    Chloramphenical 50 mg/mL 1:3333 15 μg/mL 0.15 mL

  5. Make sure to mix well after adding the antibiotic and then pour.

NaOH, 10 M Solution (for Bleach Solution)

  1. Mix the following:

    Reagent Amount for 10 mL
    NaOH 4 g
    dH2O up to 10 mL

Note: Make immediately before making bleach solution.

Bleach Solution

  1. Mix the following:

    Reagent Amount for 100 mL Amount for 200 mL
    NaOCl (from Fisher cat #SS290-1) 20 mL 40 mL
    10 M NaOH 5 mL 10 mL
    dH2O up to 100 mL up to 200 mL

  2. Store at 4°C.

2x Worm Lysis Buffer (for genotyping worms)

  1. Mix the following:

    Reagent Amount for 100 mL Final Concentrations
    KCl 0.745 g 100 mM
    Tris (pH 8.0) 0.24 g 20 mM
    MgCl2 0.047 g 5 mM
    IGEPAL 900 μL 0.9%
    Tween 20 900 μL 0.9%
    Gelatin 20 mg 0.02%

NP40/Tween are detergents, in a liquid form, so the concentration is volume per volume. Because they are viscous and difficult to pipet accurately undiluted, the easiest method is to decant some into a (graduated) 10mL conical and add an equal amount of water to get a 50% stock; mix well, and this can be pipetted accurately. Leftover 50% detergent can be saved at room temperature for a long time.

Gelatin is a solid, so should be handled as weight per volume.

  • Make 10 mL aliquots and store in the -20°C.

  • Don't try to make a 50% stock of IGEPAL - just pipette directly and be patient.

Just before use, add 20 μL 20 mg/mL proteinase K (in water, store in frozen aliquots) per mL of 2x lysis buffer.