RNAi Methods and Screening: RNAi Based High-Throughput Genetic Interaction Screening - Caenorhabditis Elegans: Molecular Genetics and Development - page 96

The bacterial clones can be re-arrayed manually into new plates by first identi-
fying the positions of the clones of interest, and then retrieving their source plates
from the 80 C freezer (place them on a bucket with dry ice to prevent thawing).
Each clone is picked with a single-channel 200 m L pipette by poking the foil that
covers the plate and dabbing the frozen bacterial culture. This culture is streaked
onto a LB agar plate with ampicillin at a concentration of 100 m g/mL and tetracy-
cline at a concentration of 12.5 m g/mL for selection. After 24 h of incubation at
37 C, an individual colony per clone is inoculated in one well of a 96-well deep well
plate containing 800 m L per well of LB broth with ampicillin at a concentration of
50 m g/mL. After 24 h of shaking the culture at 37 C and 250 rpm, 100 m L from
each well is transferred to a 96-well round bottom plate, mixed with 90% glycerol in
a 1:1 ratio, and frozen at 80 C for future use.
When complete RNAi libraries are being used as the target set, the ORFeome-RNAi
v1.1 library is provided in 96-well microtiter plates and can be used directly for the
screening in this format. The Ahringer bacterial collection is provided in 384-well
plates and must be re-arrayed in 96-well format. Each 384-well plate will result in four
96-well plates by positioning the first pin of a short 96-pin replicator tool in the wells
A1, A2, B1, and B2 of the 384-well plate, and successively ''spotting'' (not poking)
four different LB agar plates with ampicillin at a concentration of 100 m g/mL and
tetracycline at a concentration of 12.5 m g/mL (Note 1). After incubating these plates at
37 C for 24 h, the cultures can be inoculated in liquid using a long 96-pin replicator
tool, and the procedure to freeze the new plates is the same as described above (Note 2).
3. Large-Scale Worm Amplification
One way to produce a large quantity of worms from a strain maintained on
medium (60 mm 15 mm) plates is to let the worms grow until they are freshly
starved, when there are mostly L1 larvae on the plate. Then, cut out a piece of agar
(''chunk'') containing about 500 L1 larvae, and place it on a large plate. Prepare at
least 10 plates this way.
When the L1 larvae become gravid adults (about three to four days after seeding,
depending on the incubation temperature), follow the pseudo-synchronization pro-
tocol (subheading II.A.4), and dispense the resulting L1 larvae on seeded large
(100 mm 15 mm) or extra-large (150 mm 15 mm) NGM plates. In our experi-
ence, one extra-large plate corresponds to approximately 10 large plates. One extra-
large plate can be seeded with 10,000 L1 worms (Note 3).
4. Larvae Pseudo-Synchronization
We use a ''bleaching'' protocol to obtain thousands of freshly hatched and pseudo-
synchronized first-instar larvae (L1 larvae). The procedure is as follows:
1. Wash adult worms from an extra-large plate into a 50 mL conical tube, using
about 20 mL of M9 buffer (Note 4).