RNAi Methods and Screening: RNAi Based High-Throughput Genetic Interaction Screening - Caenorhabditis Elegans: Molecular Genetics and Development - page 97

2. Let the tubes with the worms settle for 10 min at the appropriate temperature (in
the 15 C incubator for most ts strains), and then aspirate the supernatant, being
careful not to aspirate the adult worms.
3. AddM9 to 10 mL, and then divide the liquid into two 15 mL conical tubes (Note 5).
Centrifuge for 2 mins at 2000 rpm and then aspirate the supernatant. The pellet in
each tube must be of similar size.
4. Add 10 mL of bleaching solution. Periodically shake the tubes and observe under
the dissecting microscope. After approximately 4 min, the adult worms start to
break apart (Note 6).
5. As soon as adult breakage begins, centrifuge for 2 min at 2000 rpm. Aspirate the
supernatant as completely and quickly as possible without disturbing the pellet.
Total contact with the bleaching solution should not exceed 10 min.
6. Add 10 mL of M9 to each tube. Invert or shake the tube to break up the pellet.
Centrifuge for 2 min at 2000 rpm, and then aspirate. Repeat this washing proce-
dure four times.
7. Resuspend in 10 mL M9 and incubate with rocking movement for 24 h (Note 7).
One extra-large plate with 10,000 gravid adults produces about 10 mL of a sus-
pension with 20-60 worms/ m L. When this suspension is diluted to 10 worms/
20 m L, it is enough to dispense L1 larvae in more than 200 96-well screening
plates.
5. Screening Protocol
Briefly, we first prepare the RNAi bacterial plates, as well as the C. elegans strains
pseudo-synchronized to the L1 stage. Using a liquid-handling robot (we use a Tecan
Aquarius TM , equipped with a carousel), the bacteria are dispensed onto plates with
flat-bottom wells. Worms are dispensed into these wells using an automated liquid
dispenser (we use a Matrix WellMate 1 ). Plates are incubated at permissive tem-
perature for three days, allowing the worms to reach the L3-L4 stage. They are then
transferred to the appropriate temperature for enhancement or suppression screen-
ing. Incubate for 3-5 days, after which the worms will have developed into adults and
produced progeny. When the worms clear the wells of bacteria, an image of each well
is captured and stored in a database for future analysis. Our protocol is shown
schematically in Fig. 3 .
Defrost Bacteria
Bacteria from source plates at -80 C are replicated to LB agar with ampicillin at a
concentration of 100 m g/mL and tetracycline at a concentration of 12.5 m g/mL on
one-well rectangular plates. Replicate the bacteria with a 96-pin tool with short pins,
using a bucket with dry ice to hold the frozen stocks. Be careful not to puncture the
agar. Incubate the plates at 37 C for 24 h, and then store at 4 C until inoculation. Do
not store plates at 4 C for more than 1 week.