RNAi Methods and Screening: RNAi Based High-Throughput Genetic Interaction Screening - Caenorhabditis Elegans: Molecular Genetics and Development - page 107

2. Wash adult worms from agar plates with M9 buffer solution and bleach them
(see larvae pseudo-synchronization protocol, subheading II.A.4). Let eggs
hatch overnight at 15 C.
Day 4
1. Count the number of L1 worms hatched per 10 m L of solution and dispense
about 50 worms into the RNAi plate previously seeded with 300 m L of bacteria.
2. Incubate the L1 larvae at 15 C until they reach the L4 stage (about 3 days).
Day 7
Move 10 L4 larvae to each of the three RNAi plates previously seeded with 30 m L
of each bacterial clone. Incubate plates at the appropriate temperature, until the L4
larvae become adults and lay eggs.
Day 9 or after
After this incubation period, remove the adult worms, and replace plates at the
appropriate temperature. After 24 h, count the number of larvae and eggs to
calculate the percentage of hatched larvae.
III. Materials
A. Reagents
1. Ahringer RNAi feeding library - Geneservice Ltd, UK ( http://www.geneservice
.co.uk/products/rnai/Celegans.jsp )
2.OrfeomeV1.1RNAifeedinglibrary-ThermoFisher,USA( http://www
.openbiosystems.com )
3. C. elegans strains and OP50 bacteria - the C. elegans Genetics Center ( http://
www.cbs.umn.edu/CGC/ )
B. Buffers and Media
1. NGM agar: 3 g NaCl, 2.5 g peptone, 17 g agar, to 973 mL with water. Sterilize
by autoclaving. Let it cool down. While it is still warm, add the following sterile
solutions: 1 mL of 1 M CaCl 2 , 1 mL of 1 M Mg SO 4 , and 25 mL of 1 M buffer
phosphate, pH 6.0. Dispense 28 mL for large plates (100 mm 15 mm) and
55 mL for extra-large plates (150 mm 15 mm).
2. Seeded extra-large NGM plates: inoculate LB broth with freshly streaked OP50.
Incubate overnight at 37 C, 250 rpm. Pellet bacteria (3500 rpm for 20 min) and
concentrate it about 25 times. Under the laminar flow chamber and using a glass
bacterial spreader, distribute 2 mL of bacterial suspension over the surface of
the NGM plate. Let dry overnight at room temperature and store at 4 C until
use.
3. M9 buffer: 5 g NaCl, 3 g KH 2 PO 4 ,6gNa 2 HPO 4 , water to 999 mL. Sterilize by
autoclaving. Add 1 mL of sterile 1 M MgSO 4 .