DNA extraction from tissue
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Protocol for extraction of DNA from tissue or embryos.
ProK 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
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
See also
External links
- DNA from fresh or frozen tissue by Ried lab
- DNA from liver by Bowtell and colleagues - cached version from protocol online
- DNA extraction from tail or tissue - at BioProtocol from unknown contributor
- Protocol Online links to DNA extraction protocols