Oneill Lab:DNA Extraction

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

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Protocol for extraction of DNA from tissue or embryos.

ProK Digestion

 * 1) Mix DNA extraction buffer
 * 2) *98 &mu;l ReagentB
 * 3) *2 &mu;l ProteinaseK
 * 4) *Mix fresh. 100 &mu;l is enough for a small pea size chunk of tissue or one embryo
 * 5) Place small piece of tissue or embryo into a microfuge tube containing 100 &mu;l of extraction buffer
 * 6) Incubate at 50&deg;C overnight

PCI

 * 1) Prepare PCI mix
 * 2) One part tris-saturated phenol to one part 24:1 Chloroform:Isoamyl alcohol
 * 3) Shake thoroughly to make emulsion
 * 4) Add one volume of PCI to extracted sample
 * 5) Shake tubes for 10 seconds
 * 6) Centrifuge at max speed for 5 minutes
 * 7) Remove aqueous phase to a new tube
 * 8) Repeat as needed
 * 9) Add one volume 24:1 chloroform:isoamyl alcohol
 * 10) Shake tubes for 10 seconds
 * 11) Centrifuge at max speed for 5 minutes
 * 12) Remove aqueous phase to a new tube
 * 13) 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 &mu;l 70% EtOH
 * 6) Centrifuge at max speed for 2 minutes
 * 7) Pipette out ethanol
 * 8) Airdry pellet
 * 9) Resuspend pellet in MilliQ water