1. Transformation of competent cells

• Thaw competent cells on ice
• Add 5µl DNA to 50µl competent cells
• Let stand on ice for >30min.
• Heat shock for 90 sec. at 42°C
• Chill on ice for >5min
• Add 50µl fresh, sterile LB to cells (for recovery)
• Incubate at 37°C for 30 min.
• Plate onto LB plates containing the appropriate antibiotics
• Incubate at 37°C overnight

2. Blunt-end ligation

• To each reaction tube add the following:

• Incubate @ 14°C for 16-24 hours
• Heat kill enzyme at 70°C for 15 min.
• Store ligations at 4°C until use

3. Preparation of competent cells

• Inoculate 5mL LB + Tet (12.5ug/mL) with a colony grown on LB + Tet plates
• Shake at 37°C overnight
• Add 400µl overnight culture to 400mL LB (1:1000 dilution)
• Shake at 180-280 RPM at 37°C for 2.5-3 hours
• Check the OD600---If not 0.3 to 0.6, shake more
• Once culture is at an appropriate OD, chill on ice for 15 min.
• Add 1 vol. (400mL) ice-cold 2x TSS
• Mix gently
• Chill on ice for 15 min.
• Pellet cells by gentle centrifugation: 1000g (2500 RPM in Sorvall with GS3 rotor)
• Check and ensure there is a pellet
• Discard the supernatant and resuspend in 0.1 vol. (40mL) 2x TSS
• Aliquot into 50-100µl aliquots
• Flash freeze samples in liquid N₂
• Store cells at -80°C until use

4. Dephosphorylation of linear plasmid DNA

• To a 0.5mL Eppendorf tube add the following:

• Incubate at 50°C for 1 hour
• Heat kill enzyme at 68°C for 20 min.
• Run on a 1% agarose gel/1x TBE
• Elute dephosphorylated vector DNA using QiaQuick gel extraction kit into 30-50µl dH₂0

5. Restriction Digest: EcoRV

• To each reaction vessel add the following:

• Incubate at 37°C for 2.5 hours
• Heat kill enzyme at 68°C for 15 min.
• Run sample on a 1% agarose gel/1xTBE
• Gel elute band using QiaQuick gel extraction kit into 30-50µl dH₂0
• Store at -20°C until use

6. Insert amplification from gDNA

• Add the following to the PCR mix:

• PCR reactions with the following program:

• Run amplified product on a 1% agarose gel/1x TBE
• Gel elute appropriate band using QiaQuick gel extraction kit into 30-50µl dH₂0
• Store at -20°C until use

7. Gel purification: QiaQuick gel purification kit

• With a clean razor, cut appropriate band(s) out of gel
• Place gel/DNA into a pre-weighed 1.5mL Eppendorf tube
• Weigh sample: should be no more than 0.4g
• Add 3 volumes (1.2mL for 0.4g sample) Buffer QG
• Incubate sample at 42°C for 10 min., vortexing occasionally
• Once gel has dissolved, check color-should be yellow
• Apply sample to a QiaQuick column and spin at full speed (13.4k RPM) for 1 min. (**The column can only take 800µl; if you have more sample add 800µl, spin, then repeat with the excess)
• Discard flow-through
• Add 0.75mL Buffer PE (w/ EtOH added) and spin for 1 min. at full speed
• Discard flow-through
• Place column in a clean, sterile 1.5 mL Eppendorf tube
• Add 30-50µl dH₂0 and let stand for 3-5 min.
• Centrifuge at full speed for 1 min.
• Keep flow-through and store at -20°C until use

8. Mini-prep

• Decant 12mL overnight culture into 12 separate 1.5mL Eppendorf tubes
• Spin at full speed (13.4k RPM) for 5 min.
• Remove supernatant and transfer pellets to 1 tube
• Spin again for 5 min. at full speed
• Remove supernatant and add 100µl Solution I; vortex
• Let stand at room temp. for 5 min.
• Add 200µl Solution II; **invert 5 times without vortexing
• Place sample on ice for >5 min.
• Add 150µl Solution III; shake tubes in rack for ~ 10 sec.
• Centrifuge at full speed for 20 min.
• Transfer supernatant to a fresh, sterile 1.5 mL Eppendorf tube
• Precipitate dsDNA with 2 volumes (1mL) 95% EtOH; vortex
• Store at -20°C for >2 hours
• Spin for 30 min. at full speed
• Remove supernatant and wash with 1 mL 70% EtOH
• Invert sample about 5 times
• Spin for 20 min. at full speed
• Remove supernatant and dry pellet in Speed Vac for 3 min. (**Be careful not to dry for too long or the pellet will be difficult to resuspend)
• Resuspend pellet in 50µl TE (pH 8.0)
• Store at -20°C until use

9. Transformant Screening

• Following transformation, pick 6 colonies from each plate (if possible) using a p1000 pipette tip
• Replica plate onto a grided 9cm LB + antibiotic plate with a small amount of the picked colony
• Incubate this plate at 37°C overnight, then place at 4°C until later use
• Place the remainder of the colony directly into PCR mix containing the appropriate primers for the insert that was ligated into the vector
• Use the following PCR format:

• Frozen glycerol stocks should be made for positive clones containing the insert of interest

10. Bacterial preparation and induction

• To NGM plates containing 25ug/ml, add 1mM IPTG to induce T7 promoter activity
• Glycerol stocked bacteria should be raised on LB plates + Amp + Tet (assuming these are the antibiotics that are required for proper selection) and then used to inoculate 100mL of LB + Amp + Tet; shake at 37°C overnight
• Streak plates with bacteria grown in LB + Amp + Tet such that there is a contiguous lawn of bacteria on the plate
• Allow plates to dry overnight (12-24 hours) at room temp. to allow the bacteria to grow and to begin induction

11. Preparation of worms for RNAi feeding

• Grow worms on OP50 prior to RNAi testing
• Bleach worms in a 25% hypochlorite, 1N NaOH solution for 3-5 min. to eliminate worm carcasses; rinse 3 times with M9 buffer
• Hatch eggs in M9 buffer overnight at room temp.
• Plate synchronized L1 larvae on NGM plates with OP50 and allow them to develop to L4 larvae (~30 hours at 25°C)
• Rinse L4 worms from plates with M9 + 0.01% Triton-X and pellet gently by centrifugation
• Wash several times with M9 + 0.01% Triton-X to remove residual bacteria
• Let stand in bacteria-free media to allow digestion of bacteria within the gut
• Plate 7-10 worms on RNAi plates and incubate for 36 hours at 22°C
• After 36 hours, transfer 3 adult worms to fresh NGM plates containing HT115(DE) bacteria expressing dsRNA
• Allow these worms to lay eggs for 24 hours, then remove them from the plate
• Plates can now be scored for embryonic lethality

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