RNAi Methods and Screening: RNAi Based High-Throughput Genetic Interaction Screening - Caenorhabditis Elegans: Molecular Genetics and Development - page 99

Amplify Worms

See subheading II. A. 3

The screening pipeline follows a 7-9 days cycle. Days 8 and 9 are image acqui-
sition days and overlap with days 2 and 3 of the following cycle ( Fig. 4 ) .

Day 1
Worm pseudo-synchronization

1. Bleach adult worms (subheading II.A.4).
2. Leave washed eggs to hatch in M9 buffer, rocking overnight at permissive
temperature.

Bacteria inoculation

1. Prepare inoculation plates by using the liquid dispensing machine
(Wellmate 1 ) to dispense 800 m L per well of LB broth with 100 m g/mL ampi-
cillin on 96-well deep well plates (Note 8). This medium is previously filtered
using with 0.22 m m membranes to maintain sterility and to avoid any potential
debris that can affect image analysis.
2. Use a sterile ''long'' 96-pin replicator tool to inoculate bacteria from the LB
agar plates into the deep wells. Cover plates with AirPore tape sheets. Shake
plates overnight at 37 C.

Day 2
Bacteria induction and re-suspension

1. Using the Wellmate 1 , add 80 m L of a 10 mM IPTG solution to each well of
bacteria culture. Re-seal the plates with AirPore tape sheets. Continue shak-
ing at 37 C for 4 h to induce the bacteria to produce double-stranded RNA.
(Note 9).
2. Centrifuge deep well plates at 3500 rpm for 5 min, and discard supernatant
(Note 10). Using the Wellmate 1 , add 300 m L of sterilized and filtrated ''com-
plete'' S-Basal medium supplemented with 100 m g/mL ampicillin, 0.1 m g/mL
Fungizone, 0.01% Tween, and 1 mM IPTG to the bacterial pellets.
3. Securely cover deep well plates with transparent plastic adhesive plate seals to
avoid cross-contamination of bacterial cultures from adjacent wells, and then
carefully vortex to re-suspend the bacteria in the S-Basal complete medium.
Continue re-suspension with multichannel pipette if necessary.
4. Using the liquid-handling robot Aquarius TM from Tecan, dispense 30 m L per
well of bacteria from the 96-well deep well plates to 96-well flat-bottom plates
(Note 11).

Worm re-suspension and seeding

5. Filter L1 worms from the bleached worm suspension with 40 m m cell strai-
ners to remove any debris, recovering the filtrate in 50 mL conical tubes
(Note 12).