Freezing C. elegans strains

  1. Grow five 6 cm plates of worms on NGM seeded with OP-50 until the plate is just starved with lots of young larvae.  The L1 larvae are the ones that survive freezing the best and are the most likely to be fertile upon thawing.  If the worms have started burrowing under the agar, it is too late, and best to start over. 

  2. Make a 1:1 mixture of M9 Buffer and 2X Freezing Buffer (FB) [2XFB is on top shelf of fridge #1, people have their own stocks of M9].  Try to maintain the sterility of the buffers when mixing.

  3. Label five CRYOSTORAGE tubes for each stock to be frozen with the strain's # and the date.  Different color capsicles fit into the tops of the cryostorage tubes.  Label both the top and the side of each tube.  It helps to write the strain number twice on the side, in case one rubs off.  Choose one color for each strain you freeze. 

  4. Wash worms from one plate to the next with the M9-FB using a transfer pipet to transfer worms and buffer.  Use a new pipet for each worm stock and be careful not to contaminate the M9-FB stock with worms.

  5. Aliquot approximately 0.5 ml volume of worm solution into each of five labeled cryostorage tubes and cap. 

  6. Put the tubes of worms into a styrofoam freezing box. Tape the outside of the box well.  Label the tape with the date and your initials.  Place on BOTTOM SHELF of the -80ºC for at least 24 hours.  You can put it anywhere in the bottom shelf of the -80ºC where you find space, usually toward the right most side. 

  7. After at least 2 days in -80ºC, Transfer tubes to liquid nitrogen tanks.

  8. Thaw a tube to check if the worms survived the freezing process.  Note, Worms must be regrown before they can be frozen again (i.e. the same worm will not survive being frozen twice). 

5% Sucrose cushion to clean up worms

This protocol should help get rid of the majority of bacteria stuck to worms. Bacteria will get stuck at the interface, whereas worms will pellet.

Use a P1000 with a barrier filter tip for the liquid handling steps. (to prevent cross contamination between strains)

  1. Wash worms off plate with 1.5 ml of EN, or M9, or 50mM NaCl and transfer to a 1.5 ml eppendorf tube.

  2. Spin 40 seconds at 2000 rpm in microfuge.

  3. Remove approximately 1200 µl of supernatant (using a P1000 with a barrier filter tip), leaving 300 µl of worms + liquid. 

  4. To a 1.5 ml screw cap tube add 1.2 ml of either (EN, or M9, or 50 mM NaCl) containing 5% sucrose.

  5. Resuspend worms in remaining 300 ul and layer over the sucrose cushion. Do not mix.

  6. Spin 40 seconds at 2000 rpm in microfuge.

  7. Remove supernatant, save worm pellet.

  8. Resuspend worm pellet in 1.5 ml either (EN, or M9, or 50 mM NaCl).  To wash off sucrose.

  9. Spin 40 seconds at 2000 rpm in microfuge

  10. Remove supernatant, save worm pellet.

  11. Flash freeze and store at -80ºC.

  12. You now have a pellet full of clean worms to use for genomic preps.

Making agar pads for C. elegans injection

Make 15 mls of 2% agarose (same kind as for DNA gels).  0.3 g in 15 mls = 2%.  Microwave carefully (don’t want to boil over).

  1. Divide into 2-3 small glass tubes (by pouring).  Keep in heat block (around 85ºC) to keep from solidifying.  Add water to an additional tube and also put in hot block.  Assemble a small glass Pasteur pipette w/ rubber bulb and place in tube with water.  The water will keep the pipette warm and prevent the agar from solidifying too quickly.

  2. Set out 12 glass coverslips (VWR custom catalog # 48368 084 30x35 -1.5 1 ounce) in sets of 2.  Prepare to be able to put 6 glass slides over them as weights (2 coverslips per slide).

  3. Pipette some hot water up and down to make sure pipette is pre-warmed, then pipette up some agarose solution.  Quickly place one drop on each slide.  Squirt out excess agar back into agarose tube and immediately pipette up and down hot water.  Place slides over agarose to flatten.  Agarose will solidify almost immediately.  Use fresh slides as needed when they become too sticky.

  4. Remove glass slides. Place coverslip with agarose pad onto metal drying rack. 

  5. Bake at 80ºC overnight in incubator under the bench #3.

  6. Agarose pad thickness will vary and depends on the amount of time before the agar drop is squished.  Ideal thickness depends on the personal preference of the experimenter.  Thicker agar dries out the worms faster, leading to faster injection time, but a shorter window of time in which to inject. 

Nextera protocol to prepare genomic DNA libraries from a C. elegans strain

 

For each Nextera reaction use 25 ng of genomic DNA. If you will be using more than that, enzyme amounts/conditions will have to be re-titrated. Set up the following reaction: 

  • 10 µl DNA+Water

  • 10 µl TDB buffer 

  • 1.5 µl Tagmentase

  • 3.5 µl H2O

  1. Mix by gently pipetting up and down the mixture, and then incubate at 55ºC for 5 minutes (or alternatively 37ºC for 30 minutes, while shaking at 350 rpm). 

  2. Once the tagmentation is done, immediately clean up with the Zymo DNA Clean and Concentrate kit, standard protocol (use 205 µl DNA binding buffer and 2x 300 µl wash buffer washes), and resuspend in 20 µl of resuspension buffer.  

  3. Set up the following PCR reaction:

  •  2 µl Tagmented DNA

  • 0.75 µl 10uM N70x primer

  • 0.75 µl 10uM N50x primer

  • 4 µl H2O

  • 7.5 µl 2x phusion

4. Use the following conditions for the PCR reaction:

  • 72ºC 3min

  • 98ºC 30sec

  • (98ºC 10sec, 55ºC 30sec, 72ºC 30sec) x 13 cycles—you may want to try 12, 13 and 14x the first time. 

  • 72ºC 5 min

5. Your library should now be amplified! Run on gel and cut out bands in 275-375 bp range.

6. Melt the PCR band with Gel purification kit buffer and clean up DNA using Zymo DNA Clean and Concentrate kit.

7. You can quantify the concentration your DNA using qubit DNA HS kit, and calculate molarity based on ~300 bp library size.

 

Worm lysis for PCR

Single worm     

  1. Prepare 15 ul aliquots lysis buffer in PCR strips. Add a single worm to each tube with lysis buffer. Check under a scope that worm is inside the tube.

  2. Place at -80ºC > 15min. Can store for several days.

  3. Incubate at 60ºC > 1 hour. Heat to 95ºC for 15 min to inactivate Proteinase K, cool to 4ºC. You can set these steps in a PCR machine.

  4. Spin briefly in a mini table top centrifuge. 

  5. Use 2.5 µl supernatant as template for PCR amplification for 25 µl reaction.   

Many worms

  1. Add 50 µl lysis buffer to tissue sample in a 0.5 ml PCR tube.  

  2. Place at -80ºC > 15min. Can store for several days.

  3. Incubate at 60ºC for 1 hour, 15 min. Make sure to vortex the tube once during this reaction.

  4. Heat to 95ºC for 15 min to inactivate Proteinase K, cool to 4ºC. You can set these steps in a PCR machine.

  5. Spin briefly in a mini table top centrifuge. 

  6. Use 1 µl supernatant as template for PCR amplification for 25 µl reaction.

 

Single Worm Nextera

Worm Digestion

  1. Pick a single worm into a drop of digestion mix (1X GC Buffer, with 2.5 ul of 20 mg/ml proteinase K per 100ul) to wash off as much bacteria as possible. Then pick worm into 15ul of digestion mix aliquoted into PCR tubes. 

  2. Incubate at 60C for 2 hours. Vortex tubes and spin down. 

  3. Bring up the volume to 100ul with ddH2O, and then clean up the DNA using Zymo’s DNA Clean and Concentrator kit following standard protocol: add 200ul of DNA binding buffer, transfer mixture to the column, centrifuge, wash the column with 200ul DNA wash buffer, then elute in 6ul of DNA Elution Buffer.  Make sure to elute into siliconized tubes since the amount of DNA from a single worm should be ≤400pg.

Nextera XT 

  1. For each Nextera XT reaction use 0.6ul of eluted DNA. If you will be using more than that, enzyme amounts/conditions will have to be re-titrated. Set up the following reaction: 10ul TDB buffer, 2ul ATM, 7.4ul H2O, and 0.6ul worm DNA. Mix by gently pipetting up and down the mixture, and then incubate at 37C for 30 minutes, while shaking at 350rpm. 

  2. While the tagmentation reaction is happening set up the PCR mix: 30ul of 2X Phusion PCR mix, 5ul of 10mM N70x, and 5ul of 10mM N50x. 

  3. Once the tagmentation reaction is done, immediately add it to the PCR mix and mix by pipetting up and down gently. 

  4. Set up the following PCR reaction: 

  • 72C 3min

  • 98C 30sec

  • (98C 10sec, 55C 30sec, 72C 30sec) x 17 cycles.

  • 72C 5 min

5. Your library should now be ready! Run on gel and cut out bands in 275-375bp range. 

 

Bleach synchronization of worms

Protocol 

  1. Prepare the following solutions: 

    • Solution 1: 2.5 mL NaOH (2N) + 1 mL Bleach (10-14%, Millipore solution kept in the fridge) + 6.5 mL Autoclaved DI water

    • Solution 2: Sterilized and filtered NaCl 50 mM (2.92 g/L)

    • Make sure you have at least 500 ml to 1 liter of Solution 2 before starting.

  2. Add 2 mL of Solution 1 to each plate and transfer eggs to the 15 mL centrifuge tubes.

  3. Make sure the total incubation time in Solution 1 is 6 minutes and 30 seconds. Bleach and NaOH activity can be variable, optimize the bleaching incubation by looking to worms in the tube under the microscope. If bleaching is not sufficient, you will end up with worm carcasses, if it’s too long your egg hatching efficiency will decrease or become zero.  

  4. Immediately after 6 min 30 sec, dilute the sample up to 15 mL with solution 2. 

    Note that the eggs must not to expose to Solution 1 for more than 6.5 minutes. (6 minutes 30 sec)

  5. Centrifuge the samples for 1000 rpm for 20 Seconds.

  6. Remove the supernatant by vacuum. Do not take too much, leave at least 2 ml of liquid when vacuuming, so that you will not lose the eggs at the bottom of the tube.

  7. Wash the eggs by adding solution 2, centrifuging and sucking out the supernatant.

  8. Repeat step 6 for two more time.

  9. Finally, remove the supernatant by pipetting and keep only 100 uL.

  10. Pipette the eggs in the new plates by pipetting remaining 100 uL and transferring them to the new plates. Make sure the liquid and the eggs are well dispersed throughout the plate.

 

Single Worm Lysis & PCR

Worm lysis

10X Worm Lysis Buffer (WLB) (Use sterile miliQ water, you can store this at room temperature ) :

  • 500 mM KCl 

  • 100 mM Tris pH 8.3

  • 25 mM MgCl2

  • 4.5% Tween-20

  1. Single worm lysis buffer (Total 100 µl):

    • 10 µl 10X WLB buffer,

    • 87.5 µl ddH2O,

    • 2.5 µl Proteinase K (20 mg/ml)

  2. Place 15 µl of lysis buffer in the bottom of a PCR tube. Aliquot single worm lysis buffer 15 µl tube per PCR tube. You can store these tubes at -80 degrees.

  3. Pick single worm (or a few worms) directly into the tube with lysis buffer + 100 µg/ml proteinase K.  As many as 10 worms can be used with this prep method.

  4. Worms can be frozen @ -80°C indefinitely.  

  5.  Set up proteinase K reaction on the PCR machine. 60°C for 60 min, 95 °C for 7 min (inactivation) 10 °C infinite. Do not keep the tubes in PCR machine overnight.

  6. You can store worm lysate reactions at -20C for future use. 

Worm PCR

  1. Make 100 uM stock of each primer in 10 mM Tris-HCl pH 8.  Dilute primers to 10 µM for PCR.

  2. Single worm PCR single reaction (20 µl total):

    • 10 µl mastermix,

    • 5.4 µl ddH2O,

    • 1 µl 10 µM Forward primer,

    • 1 µl 10 µM Reverse primer,

    • 0.6 µl DMSO

  3.  Phusion cycle:

    • Initial Denaturation: 98°C , 30 seconds 

    • Amplification (25-35 Cycles )

      98°C, 5-10 seconds
      45-72°C, 10-30 seconds
      72°C, 15-30 seconds per kb 

    • Final Extension : 72°C, 5-10 minutes 

    • Hold : 10°C 

 

C.elegans injection

  • Before hand : prepare agar pads , pull needles, prepare DNA to inhection, prepare worms 24 hrs ahead by single picking Stage L3 worms or 4 days ahead by picking and plating adult worms.

  • Prepare your injection mix.

  • Place Recovery buffer (RB ) in 65C for 10 min.

  • Prepare the injection mixture, spin down 14K for 10 min.

  • Prepare injection plate by placing kimwipes and moisturizing them by water.

  • Load 0.5 ul of injection mix to the top of the needle, roll it until you don’t see the liquid bubble at the top of the needle. Load two needles for injection.

  • Check on the microscope to see whether there are any bubbles within the injector.

  • Prepare and pick single young adult worms from seeded plates and place them to unseeded plates so that the surrounding bacteria cleans out from the worm body. Pick at least 30-40 worms for this process.

  • Stick the agarose pad on the microscope disk and put a mineral oil drop on top.

  • Load the agarose pad and the needle to the microscope. Check flow on the needle.

  • Place a single worm from the unseeded plate on the agarose pad.

  • Inject into the gonad of the worm. Pick the plane where you could see the cytoplasm between the honeycomb structure. Take a note to see the bubbling effect after the injection.

 

Small Scale C. elegans Larvae Ribo-seq

  1. Collect larvae with a pick for 2-3 hours in 50 mM NaCl.

  2. Clean up bacteria with 5% sucrose cushion.

  3. Dissolve the worms in lysis buffer: 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 5 mM MgCl2. After that, per 1 ml of solution add 1 ul 1M DTT, 10 ul 10%Triton-X, 1 ul 100 mg/ml cycloheximide. Immediately after, flash freeze in liquid nitrogen. You can store the samples at -80C at this stage.

  4. Take the frozen pellet, freeze grind it in liquid nitrogen with a pestle. Make sure it’s a fine powder.

  5. Put the powder in a tube, let it melt on ice. Add 10 ul Turbo DNAse after it melts. Divide each lysate into two for RNAseq, add trizol to the RNAseq aliquot.

  6. Vortex briefly incubate ~15 min on ice.

  7. Spin samples 10000, 5 min.

  8. OD the samples. Calculate total nanograms.

  9. Add 1.5 ul 100 U/ul RNAse1 (Ambion) per 30 ug RNA. Incubate 30 min at RT.

  10. Stop the reaction with 25 mM final VRC (Sigma made from powder, kept 250 mM stock).

  11. Load samples onto 34% sucrose made with the lysis buffer.

  12. Spin 70 000 rpm, 4 hours.

  13. Take the samples from the centrifuge. Carefully take out the supernatant. Solubilize the cushion pellet in 1 ml trizol. You can store the samples at -80C at this stage.

  14. Incubate for 5 minutes at room temperature. Add 200 ul chloroform. Spin 15k 10 min, take the aqueous layer to a new tube. Keep the trizol for later DNA precipitation.

  15. Add 50 mM 3 M NaAcetate (pH 5.5), 5 mM MgCl2, 2 ul glycoblue and 500 ul isopropanol to the sample and incubate overnight at -20C.

  16. Precipitate the Riboseq and RNAseq sample in 15K for at least 30 min. Wash the pellet in 80% ethanol. Dissolve the Riboseq pellet in 8 ul water. Dissolve the RNAseq pellet in 5 ul water+5 ul 5X FS(first strand) buffer.

  17. Add 1ul T4 PNK, 1 ul 10X T4 PNK buffer to the riboseq sample. Incubate 37C for 1 hour.

  18. Incubate the RNAseq sample for 5 min at 95C.

  19. Add equal amount of formamide dye to each sample and run 10 or 15% TBE gel (pre-run the gel for an hour). Run the gel at 200V or 20 mA fixed until the blue dye is at the bottom.

  20. Incubate the gel in TBE buffer with 1X sybr gold. Image using UV or bluebox.

  21. For the RNAseq samples, cut the bands between 35 and 50 bases. For Riboseq samples cut the bands between 26 and 34 bases.

  22. Incubate samples in 300 mM NaAcetate at 4C overnight.

  23. Precipitate the solution, add 5 mM MgCl2 total and equal volume isopropanol.

 

Staining C. elegans for ß-galactosidase activity using X-gal

by Michael Koelle, adapted from Laird Bloom, Jeff Way, & Michael Basson 4/6/94

taken from: http://www.protocol-online.org/cgi-bin/prot/view_cache.cgi?ID=970

1. Grow up a small plate of worms. Some protocols call for using ß-gal[-] bacteria on the plates (e.g. DH5 alpha) which may eliminate some staining due to bacteria in the pharynx or gut.

2. Wash the worms off the plate with ~2 ml water, transfer with a pasteur pipette to a 15 ml centrifuge tube, add water to ~5 ml, spin in a clinical centrifuge ~1 min., and remove all but ~400 ul supernatant with a pasteur pipette. Transfer the worms with a pasteur pipette to a 500 ul eppendorf tube, spin 3K for 1 min. in a variable speed microfuge, and remove as much of the supernatant as possible using a pipetteman.

3. Cap the tubes and freeze in liquid nitrogen. Open the caps and stick in a vacuum jar or speed vac for about 45 minutes to lyophilize.

4. Add ~250 ul cold acetone, and let sit in the freezer 3 min. Remove as much acetone as possible with a pipetteman, and speed vac off the rest.

5. ß-gal staining solution:

For a final volume of 1 mL, add in order:

620 ul ddH20

250 ul 0.8 M Na-phosphate buffer pH7.5

1 ul 1 M MgCl2

4 ul 1% SDS

100 ul 100 mM Redox buffer (see below, keep stocks at -20deg. C)

15 ul 5 mg/mL kanamycin (keep stock at -20deg. C)

2 ul 1 mg/ml DAPI (keep stock wrapped in foil at -20deg. C)

8 ul 5% X-Gal in dimethyl formamide. (keep at -20deg. C)

Add the X-Gal last, and then vortex quickly to avoid precipitation of the X-Gal.

Staining solution can be kept for at least 2 days (wrapped in foil at 4deg.).

Redox buffer is made fresh each time by mixing equal volumes of the following stock solutions:

100 mM Potassium Ferricyanide

100 mM Potassium Ferrocyanide

Keep both stocks at -20deg.. CAUTION: Wear gloves, these solutions are toxic (contain cyanide).

6. Add ~200 ul stain solution to the worms. Can stain at room temp or 37deg.. Periodically take out a small amount of the worm suspension, drop on a microscope slide, and examine under a high power dissecting scope to monitor progress of the staining. Some constructs require 24 hrs for the blue color to develop.

7. When the stain is satisfactory, wash the worms a couple times in PBS to remove the staining solution.

8. To view the worms, mount on an agar pad and examine on the Nomarski scope.