DNA extraction from tissue protocol

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Solutions/reagents:

• DNA extraction buffer
  
  (98µl ReagentB + 2µl ProteinaseK, mix fresh)
• 24:1 Chloroform:Isoamyl alcohol
• PCI mix
  
  (One part tris-saturated phenol to one part 24:1 Chloroform:Isoamyl alcohol, shake thoroughly to make emulsion)
• 100% EtOH
• 3M sodium acetate pH 5.0
• 70% EtOH
• MilliQ water
• small piece of tissue or embryo

Equipment:

• Incubator
• Centrifuge
• Sterile 1.5-ml microcentrifuge tubes

Steps:

1. Proteinase K digestion
   

   1. Measure out 100 µl of DNA extraction buffer into sterile 1.5-ml microcentrifuge tube (1).
      100 µl is enough for a small pea size chunk of tissue or one embryo.
      
   2. Add small piece of tissue or embryo.
      
   3. Incubate at 50°C for 12 hrs(overnight).
      

2. Phenol/chloroform/isoamyl (PCI)
   

   1. Add 1 volume PCI mix.
      
   2. Vortex the mixture for 10 secs .
      
   3. Centrifuge at maximum speed for 5 mins at room temperature and aspirate out the top layer.
      Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (2).
      Discard bottom layer.
      Repeat this step as needed.
      
   4. Add 1 volume 24:1 Chloroform:Isoamyl alcohol to sterile 1.5-ml microcentrifuge tube (2).
      
   5. Vortex the mixture for 10 secs .
      
   6. Centrifuge at maximum speed for 5 mins at room temperature and aspirate out the top layer.
      Transfer top aqueous layer into sterile 1.5-ml microcentrifuge tube (3).
      Discard bottom layer.
      

3. Ethanol precipitation
   

   1. Add 2 volumes 100% EtOH to sterile 1.5-ml microcentrifuge tube (3).
      
   2. Add 0.1 volume 3M sodium acetate pH 5.0.
      
   3. Centrifuge at maximum speed for 10 mins at room temperature, gently aspirate out the supernatant and discard it.
      
   4. Add 150 µl of 70% EtOH.
      
   5. Centrifuge at maximum speed for 2 mins at room temperature, gently aspirate out the supernatant and discard it.
      
   6. Dry the pellet in air.
      
   7. Add MilliQ water to pellet.
      Resuspend pellet by vortexing/by shaking vigorously.
      

   TOTAL TIME REQUIRED FOR THE COMPLETION OF THE PROTOCOL :~ 12 hrs, 22 mins

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