RNAi Methods and Screening: RNAi Based High-Throughput Genetic Interaction Screening - Caenorhabditis Elegans: Molecular Genetics and Development - page 108

4. S-Basal buffer: 5.85 g NaCl, 50 mL 1 M buffer phosphate pH 6.0, water to 1 L.
Sterilize by autoclaving.
5. 1 M buffer phosphate pH 6.0: For 1 L, mix 868 mL 1 M KH 2 PO 4 with 132 mL
1MK 2 HPO 4 .
6. 1 M KH 2 PO 4 :136.09 g KH 2 PO 4 , water to 1 L. Sterilize by autoclaving.
7. 1 M K 2 HPO 4 :125.4 g K 2 HPO 4 , water to 1 L. Sterilize by autoclaving.
8. S-Basal complete: To 970 mL of S-Basal buffer add: 10 mL 1 M potassium
citrate, 3 mL 1 M MgSO 4 , 3 mL 1 M CaCl 2 , and 10 mL Trace metals solution.
Sterilize by filtration. Add 1 mL of 5 mg/mL cholesterol dissolved in ethanol,
0.5 mL 100 m g/ m L ampicillin, 0.4 mL 250 m g/mL Fungizone, 1 mL 1 M IPTG,
and 1 mL 10% Tween-20. This can be prepared the day before and stored at 15 C.
9. Trace metals solution: Disodium EDTA 1.86 g, FeSO 4 .7H 2 O 0.69 g, MnCl 2
4H2O 0.20 g, ZnSO 4 7H2O 0.29 g, CuSO 4 5H 2 O 0.025 g. Dissolve in 1 L
water; aliquot into 50 mL tubes and store in dark.
10. LB broth: 5 g yeast extract, 10 g tryptone, 10 g NaCl, water to 1 L. Sterilize by
autoclaving. To grow the bacteria for the RNAi experiments, LB broth is
supplemented with ampicillin at a concentration of 50 m g/mL.
11. LB agar with ampicillin and tetracycline plates: Add 16 g/L agar to LB broth.
Sterilize by autoclaving. After it has cooled (just before pouring), add ampicillin
at a concentration of 100 m g/ m L and tetracycline at a concentration of 12.5 m g/
m L. Pour 45 mL into each 86 128 mm rectangular plate. Store at 4 C
wrapped in aluminum foil until use.
12. Bleaching solution (100 mL):4 mL 10 M NaOH, 20 mL Clorox Germicidal
Bleach (6.15% sodium hypochlorite), 76 mL autoclaved water. Prepare fresh
every time.
C. Supplies and Equipment
1. Petri dishes by Fisherbrand from Fisher
a. 60 15 mm Stackable Mono Plate (#08-757-13A)
b. 100 15 mm Stackable Mono Plate (#08-757-12)
c. 150 15 mm Slippable Mono Plate (#08-757-14)
2. OmniTray 86 128 mm rectangular plates by Nunc (#264728)
3. 96-well flat-bottom plates by Costar, Corning (#3598)
4. Matrix 96-well deep-well plates Deep Well/2 mL Blocks by Thermo Scientific
(#4222)
5. BD Falcon cell strainers, nylon mesh size: 40 m m, from Fisher
6. Stericups w/500 mL funnel, 45 mm neck size, PES membrane, Millipore filters
(#SCGP-T05-RE)
7. Fisherbrand 4oz/118 mL containers (#14-375-147)
8. AirPore tape sheets, from Quiagen (#1957)
9. Short 96-pin replicator (V&P Scientific, Inc. model VP 381)
10. Long 96-pin replicator (V&P Scientific, Inc. model VP408A)