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DNA Purification
Purifying DNA starting from a frozen C. elegans stock
Day 1: defrosting the stock
Defrost the stock into an agar plate.
Freezing a new strain of C. elegans
The strains I am working with should be isogenic but to increase the probability of working with an isogenic strain, I created
a new strain originating from a single worm. Pick a single worm into a new fresh agar NGM plate. Let it move away from the point
of deposition. Pick it again into a new fresh plate. This procedure of first picking into a plate and then into another makes sure
that no other worms or eggs are co-picked with the single worm of interest. Let the single worm populate the whole plate and
chunk the plate into 2 or more plates. This strain is now called differently, starting with SX, the code given to the Miska lab.
Freeze of of the plates.
Always freeze 5 vials. White plug in one of the 5 red caps. New strain from single worm is called
SX…. . SX is the Miska lab designation in the world. One of the tubes will be thawed tomorrow to check
that the freezing worked. One tube will be put in liquid nitrogen for long term storage. Three tubes will be
put in the -80 C freezer for mid term storage. Take a 15 ml Falcon tube, M9 medium, freezing buffer 2X.
Final volume of vial: 1 ml, so need 2.5 ml of freezing buffer. Pour 3.2 ml M9 on plate. Take 2.5 ml back with
pipetboy and 5 ml serological pipette. Put in Falcon. Add 2.5 ml freezing buffer to falcon. Pipet 1 ml of
solution in each vial. Transfer the vials into a polystyrene box. Tape the polystyrene box shut. Put the polystyrene box
in the -80 C freezer. The box allows the temperature decrease to be slower. Tomorrow, put test tube in tap water for 5 min.
Pour on plate and check that worms are alive. Put other tubes in inbox (4 tubes) and email lab manager. New strain SX…. is ready.
Day 4: pick worms into new plate
Pick a few worms into a fresh agar plate.
Day 7: bleach gravid adults
Once many gravid adults are present on the plate, bleach them and grow the eggs overnight at -20 C on a rotating wheel.
Before starting
Prepare 50 ml of bleach solution in a 50 ml Falcon tube: 4mL 10M NaOH, 3mL Sodium hypochlorite (stored in the cold room), 43mL H2O. The bleach solution can be stored at room temperature for one month.
Bleach in a tube
- Use C. elegans plates that have many gravid hermaphrodites (the eggs only will resist to the bleach treatment). Wash the plates with M9 solution: pipet M9 solution across the plate several times to loosen worms and eggs that are stuck in the bacteria. Collect the M9+worms solution in a 15 ml Falcon tube. Top up the volume to 14 ml M9.Tip: Adult hermaphrodites have a tendency to stick to the walls of plastic pipettes. To avoid this issue when harvesting the worms, you can: (i) add a small amount of detergent (NP40 0.005% or tween) to the M9 used to harvest the worms; or (ii) pre-wet the plastic pipette in M9+detergent; or (iii) use glass pipettes.
Tip: Pipette gently to avoid collecting bits of agar together with the worms.
- Wash the animals three times (or until the worm suspension is clear of bacteria) with 14 ml M9. To do so, centrifuge the animals at 2,000 rpm for 2 minutes, discard the supernatant with the vacuum pump, re-suspend the worm pellet in 14 ml M9. After the last wash, do not re-suspend the worm pellet in M9 and proceed to step 3.
- Add 4 mL of bleaching solution to the worm pellet and immediately proceed to the next step.Optional: depending on the number for worms/level of contamination, you can increase the volume of bleach solution from 4 ml to 6 ml. You can also use stronger bleaching solution (2.5mL NaOH 10M, 7.5mL Bleach, 40mL H2O). The standard conditions should work most of the time, however.
- Vortex the tube for 6 minutes. Then quickly check the mixture under a dissecting scope (through the flacon tube). The worms should be dissolved and only eggs should be visible. If so, immediately proceed to the next step.Note: If necessary, you can vortex 2 additional minutes. Do not over bleach though! If the eggs stay too long in the bleach solution, they will die.
- Add some sterile M9 to the mixture of dissolved worms/eggs in bleaching solution (up to 14 ml final volume). Spin the tube for 1 minute at 2,000 rpm to pellet the eggs. Discard the supernatant with the vacuum pump and immediately proceed to the next step.
- Resuspend the egg pellet in 14mL sterile M9. Vortex the mixture for five seconds, holding the tube horizontally. Spin the tube for 1 minute at 2,000 rpm to pellet the eggs. Discard the supernatant with the vacuum pump. Repeat this step three times.
- After the last wash, resuspend the egg pellet in the desired volume. You can either pipette the eggs onto an NGM plate (use a glass pipette) or leave the eggs in liquid suspension (200 ul of sterile M9 recommended) and place them on the rotating wheel (20°C room) overnight (you will have synchronised L1 on the next day).
Day 8: transfer L1’s onto fresh agar plate
Day 10: freeze-crack L4 worms
- Harvest the worms from the plate into a 15 ml Falcon tube. Add 16 ml of M9 + Tween (not necessarily sterile) to the plate using a serological pipette and a pipetboy.
- Pipet up and down a few times.
- Transfer the wormy solution into the 15 ml Falcon tube.
- Centrifuge the tube at 2000 rpm for 2 min.
- You should observe a worm pellet and a supernatant that does not contain swimming worms.
- Remove the supernatant using a glass Pasteur pipette inside a non-filter tip attached to a vaccum pump.
- Dissolve the worm pellet in M9 up to 14 ml. The M9 solution does not need to be sterile.
- Repeat steps 4-7 three times.
- Centrifuge the tube at 2000 rpm for 2 min.
- Remove all the supernatant as to leave 100 ul of worm pellet.
- Freeze-crack the worms in the Falcon tube in liquid nitrogen for 20 seconds holding the tube with tweezers.
- At this stage, you have the possibility to store the tube at -80 C for later use.
- Leave the cracked worm pellet to defrost at room temperature.
- Start the DNeasy kit protocol.
- Add 360 ul ATL buffer to the tube.
- Transfer the tube to a 1.5 ml eppendorf tube.
- Add 40 ul proteinase K to the tube.
- Leave the tube overnight on a shaker (550 rpm) at 56 C.
Day 11: purify DNA
Centrifugation to get rid of debris + DNeasy protocol
- Centrifuge at 8000 g for 1.5 min.
- Transfer the supernatant to a new tube
- add 4 ul RNAse A
- vortex a bit
- leave 2 min at RT
- vortex
- add 400 ul AL
- vortex
- add 400 ul EtOh to tube.
- Vortex.
- load on column
- centrifuge 8000 g for 1 min
- discard flow-through and tube
- place column onto new tube
- add 500 ul AW1
- centrifuge 1 min at 8000 g
- discard flow-through and tube
- place column into new collection tube
- add 500 ul AW2
- centrifuge 3 min at 20,000 g
- discard flow-through and collection tube
- centrifuge 1 min at 20,000 g
- discard flow-through and collection tube
- place column into 1.5 ml tube
- add 100 100 mM Tris pH 8, leave 1 min at RT
- centrifuge 1 min at 8000 g
Nanodrop
Measure the nucleic acid concentration using Nanodrop.
Lift pedestal.
Clean sample area.
Drop pedestal.
Touch screen.
-> Nucleic Acids -> dsDNA
Instrument self-test…
Lift pedestal.
Clean pedestal.
Load blank.
Lower arm.
Export Nanodrop results onto USB stick.
Qubit
Measure the dsDNA concentration using Qubit.
Use low sensitivity dye (BR: broad range) dilute 200x reagent in buffer to give working solution prepare two
standards tubes in Qubit tubes
- standard 1: 190 ul working solution + 10 ul standard aliquot
- standard 2: 190 ul working solution + 10 ul standard aliquot
- sample: 190 ul working solution + 10 ul sample
Qubit results for sample: X ng/ul
Export Qubit results onto USB stick.
Agarose gel
Agarose gel: 250 ml flask. Add 0.45 g agarose in 60 ml TAE in measuring cylinder.
Tape container. Put comb in container. Microwave 1 min. Add 0.6 ul ETBR to solution.
Stir. Pour in 4 C room. Add water in flask. Stir. Pour through white material for ETBR. Wash flask with water.
Remove tape. Put gel in container. Add 5 ul ladder. Add 2 ul loading buffer to 5 ul DNA sample and load into gel.
Turn on machine on right. 150 mV, 400 mA, 30 min. Check run every 10 minutes.
Put gel in Imager. Open Image Lab. New Protocol.
Position gel, zoom in/out. Start Protocol. Image is ready on computer.
Invert contrast, change contrast, print image. Don’t touch the computer without gloves. It is contaminated.
Thanks to Jérémie Le Pen and others from the Miska lab for the invaluable advice.