DNA extraction and precipitation with phenol-chloroform
The process is divided into three phases: homogenisation, extraction, and resuspension.
The first step is to break open the cells and suspend their contents in an extractable form.
- If your sample is cells in liquid (e.g. blood, spit), pelletise the sample by centrifuging. Discard the liquid.
- Suspend in 100ul homogenisation buffer, comprising 10mm edta, 60mm nacl, and 10mm tris (lowered to a pH of 7.5 using HCl)
- Add equal volume solubilisation buffer, comprising 2% SDS, 30mm EDTA, 200mm tris (lowered to a pH of 9 using HCl)
- Add 5ul/ml proteinase-k (i.e. 10 ul)
- Incubate at 37C for 1 hour
- Add equal volume phenol-chloroform (i.e. 200ul)
- Emulsify by shaking (gently) or upending.
- Centrifuge at 13000 RPM for 10 minutes.
- You should see an organic layer at the bottom, an interface layer, and an aqueous layer at the top. Take the aqueous layer.
Precipitation and resuspension
- Add 0.1 volumes sodium acetate (i.e. for 100ul of supernatant, add 10ul sodium acetate)
- Add 0.7 volumes IPA.
- Incubate at room temperature for 10-15 minutes
- Spin down at 13000 RPM for 10-15 minutes.
- Decant the IPA leaving only a glassy / clear pellet at the bottom. (IPA-precipitated pellets are glassy / clear; ethanol-precipitated pellets are white)
- Let the pellet dry completely so that no IPA remains, perhaps by incubating at 37C.
- Resuspend to 100ul with TE buffer, nuclease-free water, or R0 water.