Proteinase K digestion
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
Place small piece of tissue or embryo into a microfuge tube containing 100 μl of extraction buffer
Incubate at 50°C overnight
Phenol/chloroform/isoamyl (PCI)
Prepare PCI mix
One part tris-saturated phenol to one part 24:1 Chloroform:Isoamyl alcohol
Shake thoroughly to make emulsion
Add one volume of PCI to extracted sample
Shake tubes for 10 seconds
Centrifuge at max speed for 5 minutes
Remove aqueous phase to a new tube
Repeat as needed
Add one volume 24:1 chloroform:isoamyl alcohol
Shake tubes for 10 seconds
Centrifuge at max speed for 5 minutes
Remove aqueous phase to a new tube
Continue to precipitation
Ethanol precipitation
Add 2 volumes 100% EtOH
Add 1/10 volume 3M Sodium Acetate pH 5.0
Centrifuge at max speed for 10 minutes
Decant ethanol
Add 150 μl 70% EtOH
Centrifuge at max speed for 2 minutes
Pipette out ethanol
Airdry pellet
Resuspend pellet in MilliQ water
