Protocol used for PrepGEM digestion of individual small nematodes in 0.5 ml tubes:
- Label the tops and sides of 0.5ml PCR tubes with the appropriate individual nematode number (e.g., R001, R002, etc.).
- Prepare 300 uL of 1X PrepGEM Gold buffer using PCR grade water. Use a 1.7ml tube for this solution. This is the solution used for cutting the nematodes and transferring them to the digestion tubes.
- Prepare a digestion mix consisting of: 21.25 uL PCR grade water and 2.5uL PrepGEM Gold 10X Buffer per digestion.
- Aliquot 23.75 uL of digestion mix into each 0.5 ml PCR tube. Keep these tubes refrigerator prior to use.
- Transfer the nematode (e.g., following microscopy) to a drop of buffer prepared in step 2 above. Cut the nematode in two using a tuberculin needle and syringe. Use a P2 pipetter to draw up the nematode pieces in about 1 uL of the buffer and place in the appropriate labeled tube containing digestion mix as aliquoted in step 4.
- Use the dissecting microscope to visually confirm the presence of nematodes in each tube following transfer. Use a microcentrifuge to collect all fluid into the bottom of tubes as needed.
- Store the tubes from step 6 in the -20 C freezer until ready to treat a group of individual samples.
- When treating a group of samples: Add 0.25 uL of PrepGEM enzyme to each tube using a P-2 pipettor and separate tips for each tube. Gently mix tube using your finger to “vortex” the contents. Spin the tube down briefly in a microcentrifuge to collect all fluid into the bottom as needed.
- Incubate tubes @75C for 1 hour in a thermal cycler. Set thermal cycler to use heated lid.
- Remove the tubes from the thermal cycler. Freeze/thaw the tubes 3 times using dry ice. Samples will freeze in ~4 min. Spin the tube down briefly in a microcentrifuge to collect all fluid into the bottom as needed.
- Return tubes to the thermal cycler and incubate again @75C for 1 hour. Set thermal cycler to use heated lid.
- Heat kill the PrepGEM enzyme at 95C for 10 min. Set thermal cycler to use heated lid.
- Freeze the tubes at -20C for future use in PCR reactions.
Protocol used for extracting DNA from individual nematodes and their associated bacterial DNA
- Pick individual nematodes from culture, or if from soil, isolated using sucrose centrifugation.
- Wash living nematodes using solution A (~400 ul). Spin at 7,000 rpm in the Eppendorf centrifuge for 2 min. Rinse nematodes from tube using solution A (see end for solution composition).
- Place nematodes in a clean depression slide in solution A. Heat kill at 75C using PCR machine block (turn heated lid off) or dry block.
- Wash nematodes 2x by vortex/centrifugation in solution A (use 0.5 ml tube). Spin at 7,000 rpm in the Eppendorf centrifuge for 2 min each wash.
- Place nematodes in a small petri dish in solution A. Rinse nematodes from tube using solution A.
- Use P2 pipettor to move individual nematodes from the original petri dish into a new petri dish containing about 200 ul of solution A.
- Cut nematode using a tuberculin syringe/needle. Use a new needle/syringe for each nematode. Dispose of syringe properly (do not recap).
- Use a P2 pipettor to transfer the nematode pieces to a 0.5ml tube containing 50ul of Tissue and Cell Lysis solution (Epicentre kit). Set P2 to 1 ul for each transfer (2ul total). Express nematodes from tip and recheck tube or tip for pieces. Freeze/thaw the tubes on dry ice 3 or more times.
- Add 1 ul of proteinase K (20 mg/ml). Incubate tube at 65 C for 1 hr in PCR machine with heated lid. Check for progress of nematode digestion. Continue 65 C incubation as needed to complete digestion. When digested, heat kill at 95 C for 10 minutes using PCR machine and heated lid.
- Add 2 ul of lysozyme (10mg/ml) to each tube and incubate at 37 C for 30 minutes. Heat kill lysozyme at 95 C for 10 minutes.
- Add 1 ul of proteinase K (20 mg/ml). Incubate tube at 65 C for 1 hr in PCR machine with heated lid. Heat kill at 95 C for 10 minutes using PCR machine and heated lid.
- Add 30 ul of protein precipitation reagent (Epicentre kit). Vortex. Centrifuge for 10 minutes.
- Transfer supernatant from step 12 into new 1.5 or 1.7 ml tube. Add 7 ul of polyacryl carrier. Add 200 ul of 2-propanol (isopropanol). Mix contents. Precipitate DNA with overnight incubation in -20 freezer.
- Centrifuge at maximum rpm for 15 minutes to recover pellet. Wash pellet two times with 70% ethanol. Dry “pellet” and re-suspend in 10-15 ul lambda TE buffer.
Solution A – 0.05% Triton X-100 in PCR grade water (12.5 ul in 25 ml Molecular Biology Grade water)
Lysozyme solution – 10 mg/ml in 100 mM Tris-HCl pH 8. Aliquot and freeze.
Chelex DNA extraction of single nematodes
Need: Chelex resin (Sigma sells this as “Chelex 100”), Proteinase K, ultrapure water.
- Make a 5% Chelex soln (e.g. for a 10mL solution)
- A) place stir bar in 50mL conical tube held upright in a beaker
- B) add 0.5g Chelex resin into conical tube
- C) fill to 10 mL with sterile water (this solution can be stored in the refrigerator for up to 1 month).
- Use a sterile razor blade to cut the end off of a P1000 tip to make the opening bigger (Chelex beads are too big to fit otherwise).
- With stir bar, mix up chelex soln (make sure chelex beads are spinning vigorously in the water) and take 20uL and add to 0.5 ml tubes (make sure some chelex beads are present in each tube).
- Add 1 uL of proteinase K solution (20mg/mL) to each tube.
- Cutting each in half with a tuberculin needle or other device and add nematodes to tube.
- Incubate at 56 C for 1 hour.
- Boil at 100 C for 8 min (100 C using a thermocycler).
- Cool down for a few seconds (if using thermocycler bring to 40 C for about 30 sec).
- Vortex for about 30 seconds.
- Use 4 uL or less per PCR reaction, making sure to leave the beads behind.
NaOH digestion of single nematodes
From Floyd, Abebe, Papert, Blaxter: Molecular Ecology April 2002.
- Put one nematode in 20 uL of 0.25 M NaOH (this must be freshly prepared for each day the extractions are done; typically 1 NaOH pellet in 11.6 ml water).
- Spin down to ensure nematode is fully covered in solution.
- Optional step: freeze/thaw on dry ice or at –80 C.
- Incubate overnight at 25 C.
- Heat 3 minutes at 99 C (PCR machine).
- Cool to room temperature.
- Spin down to collect any liquid on side of tube.
- Add: 4 uL 1 M HCl, 10 uL 0.5 M Tris-HCl (pH 8.0), 5 uL 2% Triton X-100
Mix briefly and spin.
- Heat 3 minutes at 99 C.
- Cool to room temperature and store at –80 C.
- pH of digest should be between 8 and 9.
- Use 1-2 uL per 25 uL PCR reaction.
DNAzol kit DNA isolation protocol for individual or pooled nematodes
- Place each live (preferably) nematodes in 100 uL digestion solution in a 0.5 ml Phoenix tube
100 mM Tris HCL pH 7.6 200 uL
200 mM NaCl 200 uL
0.5 M EDTA pH 8.0 400 uL
10 % Sarkosyl 200 uL
Proteinase K (10 mg/ml) 20 uL
Ultrapure water 980 uL
Note: Proteinase K should be made fresh – usually 0.005 g/0.5 ml
- Leave at room temperature for at least 0.5 hour to allow worm to digest solution.
- Digest overnight in 56 °C water bath.
- Heat kill proteinase with 95 °C program in PCR machine (15 minutes).
- Freeze and then thaw tubes 4 times.
- Centrifuge sample for 5 minutes at 10,000 rpm.
- Remove 95 uL of solution, leaving bottom 5 mL, and add it to 1 ml of DNAzol isolation reagent in a 1.7 ml tube.
- Add 6 uL Polyacryl Carrier solution, vortexing Carrier first.
- Mix tube by inverting 5 times.
- 10 Add 0.5 ml 100 % ethanol and mix by inverting 10 times
- Let sample sit at room temperature for 5 minutes
- Pellet DNA by centrifuging at 7,000 rpm for 5 minutes
- Pour off ethanol and then wash DNA twice with 800 uL 75% ethanol, spinning again if pellet breaks loose
- Pour off final ethanol, then remove last of it with a pipet.
- Allow visible ethanol to evaporate but do not allow pellet to dry.
- Resuspend in 6-20 uL TE, depending on how many nematodes you started with (less TE for fewer nematodes to make the DNA more concentrated).