(last update : 15-05-2009)

gDNA extraction

gDNA extraction of a few worms with WLB

  1. Reagents needed :

    2. WLB : Worm lysis buffer :

  2. Method 1 : cutting the worm in pieces

    1. bring one or a few worms in 20 µl WLB on a sterile microscope slide and cut them in two
    2. pipet the WLB with the worm pieces into a 0,5 ml tube
    3. freeze 10' at -80°C
    4. incubate at 65 °C for 1 hour followed by 10 minutes at 95°C
    5. centrifuge 1 minute at maximum speed
    6. use 1 - 5 µl for PCR

  3. Method 2 : beadbeating

    1. transfer one or a few worms in 30 µl WLB in a 0,5 ml tube
    2. freeze 10' at -80°C
    3. add 50 mg glasbeads and beadbeat the tube 30 seconds at maximum speed
    4. spin down
    5. incubate at 65 °C for 1 hour followed by 10 minutes at 95°C
    6. centrifuge 1 minute at maximum speed
    7. use 1 - 5 µl for PCR

  4. Method 3 : if you have one or a few nematodes in a 0,5 ml tube with aceton (without other organic material or debris)

    1. add 30 µl WLB to the aceton in the tube
    2. dry the sample under vacuum
    3. add 30 µl sterile water to the tube and 1 µl Proteinase K (because the salt from the WLB is still in the tube, this results in 30 µl WLB)
    4. mix and spin down
    5. incubate at 65 °C for 1 hour followed by 10 minutes at 95°C
    6. centrifuge 1 minute at maximum speed
    7. use 1 - 5 µl for PCR

Extraction of gDNA from a few worms with CTAB

  1. Reagents needed :

  2. Method :

    1. transfer a few nematodes in a 0,5 ml tube in 5 µl destilled water
    2. add 100 µl CTAB and freeze 10 minutes at -80°C
    3. add ± 0,05 g glass beads and 1,2 µl proteinase K (1 mg/100 µl)
    4. beadbeat during 30 seconds at 5000 cycles/minute (if the ice is almost completely defrosted)
    5. incubate 30 minutes at 60°C shake regularly
    6. add 100 µl chloroform/isoamylalcohol (24/1) and shake gently
    7. centrifuge 10 minutes at 8.000 rpm (4°C)
    8. transfer the aqueous fase into an other tube and add 1 µl glycogen and an equal volume cold isopropanol
    9. put 1 hour at roomtemperature
    10. centrifuge 20 minutes at 14.000 rpm (4°C)
    11. remove supernatans
    12. add 250 µl washing buffer and leave 10 minutes on ice
    13. centrifuge 10 minutes at 14.000 rpm (4°C)
    14. remove supernatans and dry under vacuum (not to long, otherwise the DNA will not dissolve in the next step. An other possibility is to spin down the remaining drops of ethanol and suck it out with a fine needle
    15. add 20 µl sterile water
    16. use 1 to 5 µl for PCR

Extraction of gDNA from a lot of nematodes (from cultures) with CTAB

The nematodes can be cleaned with sucrose flotation if necessary.
  1. Reagents needed :

  2. Method :

    1. rince the nematodes from the agar plate with2 x 1 ml sterile S-buffer and transfer them into a 2 ml tube with screwcap
    2. put on ice for a few minutes an centrifuge for 3 minutes at 3.000 rpm
    3. remove supernatans
    4. add 500 µl CTAB and freeze 10 minutes at -80°C
    5. add ± 0,1 g glasbeads and 6 µl proteinase K (1 mg/100 µl)
    6. beadbeate during 30 seconds at 5000 cycles/minute (if the ice is almost completely defrosted)
    7. incubate 30 minutes to 1 hour at 60°C and shake regularly
    8. add 250 µl ammonium acetate 7,5 M and shake gently
    9. centrifuge 10 minutes at 14.000 rpm (roomtemperature !)
    10. transfer the aqueous fase into an other tube (pellet is precipitated protein) and add an equal volume cold isopropanol and shake gently
    11. put 1 hour at roomtemperature
    12. centrifuge 15 minutes at 14.000 rpm (4°C)
    13. remove supernatans
    14. add 1 ml washing buffer and leave 30 minutes on ice
    15. centrifuge 5 minutes at 14.000 rpm (4°C)
    16. remove supernatans and dry under vacuum (not to long, otherwise the DNA will not dissolve in the next step. An other possibility is to spin down the remaining drops of ethanol and suck it out with a fine needle
    17. add 20 µl sterile water
    18. use 1 to 5 µl for PCR

Extraction of gDNA from Crustaceae with CTAB

  1. Reagents needed :

  2. Method :

    1. if you use fresh animals, just rince it with destilled water, or if it was fixated in Carnoy solution, rince it twice with destilled water and leave it 1 hour in destilled water (to extract the Carnoy solution)
    2. bring the animal or some muscular tissue in a 2 ml tube with screwcap
    3. add 500 µl CTAB and freeze 10 minutes at -80°C
    4. add ± 0,1 g glasbeads and 6 µl proteinase K (1 mg/100 µl)
    5. beadbeat during 30 seconds at 5000 cycles/minute (if the ice is almost completely defrosted)
    6. incubate 2 hours at 60°C and shake regularly. If the tissue is not completely dissolved, add 3 µl proteinase K and incubate overnight at 37°C)
    7. add an 250 µl ammonium acetate 7,5 M and shake gently
    8. centrifuge 10 minutes at 14.000 rpm (roomtemperature !)
    9. transfer the aqueous fase into an other tube (pellet is precipitated protein) and add an equal volume cold isopropanol and mix
    10. put 1 hour at roomtemperature
    11. centrifuge 15 minutes at 14.000 rpm (4°C)
    12. remove supernatans
    13. resuspend the pellet in 30 µl sterile water and add 1 µl RNAse
    14. incubate 15 minutes at 60°C
    15. add 1 µl glycogen if you started from a small animal and add an equal volume cold isopropyl alcohol and mix
    16. put 1 hour at roomtemperature
    17. centrifuge 15 minutes at 14.000 rpm (4°C)
    18. remove supernatans
    19. add 1 ml washing buffer and leave 30 minutes on ice
    20. centrifuge 5 minutes at 14.000 rpm (4°C)
    21. remove supernatans and dry under vacuum (not to long, otherwise the DNA will not dissolve in the next step. An other possibility is to spin down the remaining drops of ethanol and suck it out with a fine needle)
    22. add 30 µl sterile water
    23. use 1 to 5 µl from serial dilutions for PCR

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