Stickleback

Isolating Stickleback DNA (Phenol-Chlorofom-Isoamyl-Alcohol Based Protocol)

Modified by Antoine - Oct. 2015

Day 1: Tissue collection and digestion

  1. Preparation:

    1. Turn on water bath, make sure it is set to 55ºC.

    2. Aliquot 600 μl of tissue digestion buffer*1 into appropriate number of 1.5 ml tubes (1 tube per specimen).

    3. Make specimen labels if specimens are to be preserved.

  2. Cut off caudal fin near base and the right pectoral fin (be extremely careful not to damage the caudal peduncle, we may want to count plates and measure SL).

  3. Place the fin clips into tube with 600 μl of tissue digestion buffer.

  4. Add 10 μl of proteinase K2.

  5. Vortex briefly and place tubes in 55ºC water bath overnight.

Day 2: DNA Isolation – Step 1

  1. Vortex briefly. Remember to turn off water bath.

  2. Take samples to hood. Add 600 µl of 25:24:1 phenol:chloroform:isoamyl alcohol solution3 (this solution is highly toxic and tends to drip from the pipette, so handle with care).

  3. Shake in hood for ~ 3mn until completely mixed (hold tops of tubes, otherwise they may pop open!). Solution should look milky white (it is important to mix completely).

    1. Spin 10 minutes at room temperature at maximum speed 15,000 rpm.

    2. When complete, gently remove tubes from centrifuge and take them to hood.

    1. In hood, carefully remove (top) layer into fresh tube (appropriately labeled) 1.5 ml tubes with 1000µl of COLD (-20°c) 100% ETOH. Use P200 to remove top layer. You should be able to get ~ 450µl max.

    2. This is a good time to change gloves and tube rack.

  6. Mix by inverting several times.

  7. Place at -20°c overnight to let DNA precipitate

Day 3: DNA Isolation – Step 2

  1. Spin 10 minutes at room temperature at 12,100 rpm.

  2. After centrifugation you should see a gray/white pellet at the bottom of the tube. Remove ethanol without disturbing the pellet.

  3. Add 500 μl of freshly prepared 70% (at room temperature) ethanol5.

  4. Spin 5 minutes at room temperature at 12,100 rpm.

  5. Remove 70% ethanol carefully with a pipette (use P200 to avoid disturbing the pellet).

  6. Spin briefly and remove as much of the residual ethanol as possible (otherwise ethanol may interfere with PCR).

  7. Dry pellet for 15 minutes at room temperature.

  8. Resuspend (pipette up and down several times to dissolve the pellet) in 100 μl of TE6, this is concentrated stock (store at -80ºC if needed ).

  9. Quantify with Nanoquant or PicoGreen for dsDNA ratio.

  10. Working stock for PCR reactions is 1:25 dilution- 5 μl concentrated stock + 120 μl H2O. Use PCR tube racks and store at -20ºC. Concentrated stock is ideal for ddRAD library prep.

Solutions

    1. Tissue digestion Buffer:

      • 10 mM Tris, pH 8.0

      • 100 mM NaCl

      • 10 mM EDTA

      • 0.5% SDS

    2. Combine the following to make 500 ml:

      • dH2O 450 ml

      • 1 M Tris, pH 8.0 5 ml

      • 5 M NaCl 10 ml

      • 0.5 M EDTA 10 ml

      • 10% SDS 25 ml

    3. Notes on making stock solutions:

    Reagent

    MW/FW

    M

    For 250 mL

    Comments

    SDS

    (Comes in 20% Solution)

    Tris

    121.1

    1

    30.275 g

    Adjust pH with HCl

    NaCl

    58.44

    5

    73.05 g

    EDTA*

    372.24

    0.5

    46.53 g

    *Will not dissolve if pH is below 8. Adjust pH with Sodium hydroxide pellets as you mix on stirring plate

  2. Proteinase K (20 mg/ml, comes in solution)

  3. 25:24:1 Phenol:chloroform:isoamyl:alcohol solution (or 1:1 phenol chloroform solution).

  4. 100% ethanol

  5. 70% ethanol

  6. TE: Tris-EDTA (10 mM Tris pH 8, 1 mM EDTA pH8). To make 250 ml combine 2.5 ml 1 M Tris, 0.5 ml of 0.5M EDTA, and 247 ml H2O.