DNA extraction from tissue

Proteinase K digestion

1. Mix DNA extraction buffer
   • 98 μl ReagentB
   • 2 μl ProteinaseK
   • Mix fresh. 100 μl is enough for a small pea size chunk of tissue or one embryo
2. Place small piece of tissue or embryo into a microfuge tube containing 100 μl of extraction buffer
3. Incubate at 50°C overnight

Phenol/chloroform/isoamyl (PCI)

1. Prepare PCI mix
   1. One part tris-saturated phenol to one part 24:1 Chloroform:Isoamyl alcohol
   2. Shake thoroughly to make emulsion
2. Add one volume of PCI to extracted sample
3. Shake tubes for 10 seconds
4. Centrifuge at max speed for 5 minutes
5. Remove aqueous phase to a new tube
6. Repeat as needed
7. Add one volume 24:1 chloroform:isoamyl alcohol
8. Shake tubes for 10 seconds
9. Centrifuge at max speed for 5 minutes
10. Remove aqueous phase to a new tube
11. Continue to precipitation

Ethanol precipitation

1. Add 2 volumes 100% EtOH
2. Add 1/10 volume 3M Sodium Acetate pH 5.0
3. Centrifuge at max speed for 10 minutes
4. Decant ethanol
5. Add 150 μl 70% EtOH
6. Centrifuge at max speed for 2 minutes
7. Pipette out ethanol
8. Airdry pellet
9. Resuspend pellet in MilliQ water