DNA extraction from tissue-PDF
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 a small piece of tissue or embryo into a microfuge tube containing 100 μl of extraction buffer
- Incubate at 50°C overnight
- 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 the extracted sample
- Shake tubes for 10 seconds
- Centrifuge at max speed for 5 minutes
- Remove the aqueous phase to a new tube
- Repeat as needed
- Add one volume of 24:1 chloroform: isoamyl alcohol
- Shake tubes for 10 seconds
- Centrifuge at max speed for 5 minutes
- Remove the 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