Oneill Lab:DNA Extraction

Michael J. O'Neill Lab University of Connecticut Department of Molecular and Cell Biology

Back to O'Neill Lab Protocols

Protocol for extraction of DNA from tissue or embryos.

ProK 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

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