Extraction of genomic DNA from C.elegans using Qiagen kit

1. Collect worms from one 60mm/ two 35mm petri plates in dH2O or M9 buffer and transfer to 15ml tube.
2. Pellet down the worms by centrifugation at 2500 rpm at room temperature for 30 seconds.
3. Repeat steps 1 and 2 twice.
4. Wash with M9 buffer/dH2O twice.
5. Remove the supernatant and re-suspend the worms in 200ul of ATL buffer (Kit).
6. Transfer the worms to a 1.5ml Eppendorf tube. Incubate at -80 ᴼC overnight

(Worms can be stored indefinitely at -80ᴼC)

1. Follow 3 cycles of Freeze (-20ᴼC)/thaw (37ᴼC).
2. Add 20ul of Proteinase K (Kit) and incubate at 56ᴼC for 3 hours. Vortex in between.
3. (Optional step)Add 8ul of RNAse A (not in Kit) (50 mg/ml) and incubate at room temperature for 5 min.
4. Add 200ul of AL buffer (Kit) and mix/vortex. Incubate for 10 min at 56ᴼC.
5. Add 200ul of ethanol (100%) (not in Kit)  and mix/vortex.
6. Pipet the mixture into a DNeasy Mini spin column placed in a 2ml collection tube (KIT). Centrifuge at 8,000rpm for 1 min at RT. Discard flow-through.
7. Add 500ul AW1 buffer (Kit). Centrifuge at 8,000rpm for 1 min at RT. Discard flow-through.
8. Add 500ul AW2 buffer (Kit). Centrifuge at 14,000rpm) for 3min to dry the column membrane. Discard flow-through and collection tube. Spin for 2 min to get rid of wash buffer AW2
9. Place the column in a 1.5 ml Eppendorf tube and pipet 50ul of sterile dH2O/ AE buffer (Kit) (or 1X TE) directly in the middle of the membrane. Incubate at room temperature for 1 min and then centrifuge for 1 min at 8,000 rpm at RT to elute.
10. Repeat the step 15 by adding another 50ul of sterile dH2O/ AE buffer (or 1X TE) directly onto the membrane in the same Eppendorf tube.
11. Check quality and quantity of DNA on agarose gel. This procedure yields around 20-30ng/ul of DNA.
12. Store the DNA at 4ᴼC no freezing required, it is good for PCR application.

DNeasy Blood and Tissue kit cat. No 69504 and 69506, kept at RT. 

Seeding of OP50 bacterial culture on the NGM plates

 DAY 1 (afternoon)

1. OP50 requirements: 50ul for 35mm plate, and 100ul for 60mm plates.
2. Inoculation of OP50 in the desired volume of LB.
3. Grow overnight at 37oC with agitation at 220 RPM.

DAY 2 (next morning)

1. Using sterile tips, pipette 50/100ul of the OP50 culture in the center of a NGM plate.
2. Spread the drop evenly, without reaching to the edge of the plate (see below).
3. Dry them at RT for 30-60 minutes. Can be stored at RT or 4oC in air tight packet.

 
Short cut Bleaching C. elegans

Bleach Solution (freshly pepared)
500 ul of 1 N NaOH
416 ul of 6% bleach
80 ul  sdw

1 ml bleach solution.
Add a drop at seeded plate in the corner 
Put worms in the drop
Worm open up and release eggs

Decontamination or Bleaching of worms

Requirements:

• NaOH 10N or KOH 5M
• Bleach (Sodium hypochlorite)
• NGM plates (with OP50)
• ddH2O/M9 sterile/filtered

Protocol

1. Collect worms from the plates in ddH2O/M9 buffer and transfer to a 15ml tube. Let the worms settle. Spin at 2500rpm for 1 min, Repeat 3 times with ddH2O/M9 buffer.
2. Transfer to 1.5 ml tube in 3.75ml of ddH2O or M9.
3. NaOH/bleach solution must be prepared fresh
4. Mix 0.25ml of NaOH 10N or 500 ul of 5M KOH with 1ml of bleach. Add to worm solution. Vortex briefly.
5. Incubate at room temperature until worms are no longer visible or moving with the naked eye (usually 4-6 minutes). Vortex briefly every minute. Not beyond 10 min.
6. Centrifuge 1min at 1200g. Remove supernatant. Wash with M9 buffer
7. Repeat step 3-5 times.
8. After the last wash, leave approx. 100 ul above the egg pellet in M9 buffer.
9. Pipette drop of it at the side of a NGM petri, Leave at RT for 15 min to dry up
10. Grow it overnight at 20oC.