Stickleback
============

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

Modified by Antoine - Oct. 2015


Day 1: Tissue collection and digestion
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#. Preparation:
 
   a. Turn on water bath, make sure it is set to 55ºC.
   b. Aliquot 600 μl of tissue digestion buffer*1 into appropriate number of 1.5 ml tubes (1 tube per specimen). 
   c. Make specimen labels if specimens are to be preserved.

#. 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).
#. Place the fin clips into tube with 600 μl of tissue digestion buffer.
#. Add 10 μl of proteinase K2.
#. Vortex briefly and place tubes in 55ºC water bath overnight.


Day 2: DNA Isolation – Step 1
--------------------------------
#. Vortex briefly. Remember to turn off water bath.
#. 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).

#. 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).

#.  
   a. Spin 10 minutes at room temperature at maximum speed 15,000 rpm.
   b. When complete, gently remove tubes from centrifuge and take them to hood.   

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

#. Mix by inverting several times.
#. Place at -20°c overnight to let DNA precipitate

Day 3: DNA Isolation – Step 2
---------------------------------
#. Spin 10 minutes at room temperature at 12,100 rpm.
#. After centrifugation you should see a gray/white pellet at the bottom of the tube. Remove ethanol without disturbing the pellet.
#. Add 500 μl of freshly prepared 70% (at room temperature) ethanol5.
#. Spin 5 minutes at room temperature at 12,100 rpm.
#. Remove 70% ethanol carefully with a pipette (use P200 to avoid disturbing the pellet). 
#. Spin briefly and remove as much of the residual ethanol as possible (otherwise ethanol may interfere with PCR).
#. Dry pellet for 15 minutes at room temperature.
#. 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 ).
#. Quantify with Nanoquant or PicoGreen for dsDNA ratio.
#. 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
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#. a. Tissue digestion Buffer:
	*  10 mM Tris, pH 8.0
	*  100 mM NaCl
	*  10 mM EDTA
	*  0.5% SDS

   b. 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

   c. **Notes on making stock solutions:**

   .. csv-table::
      :header: "Reagent", "MW/FW", "M", "For 250 mL", "Comments"
      :widths: 5, 5, 5, 5, 15

      "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"

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

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

#. 100% ethanol

#. 70% ethanol

#. 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.