DNA extraction and precipitation with phenol-chloroform

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--Wzdd (talk) 21:11, 2 October 2016 (BST)== Phenol-chloroform extraction ==

The process is divided into three phases: homogenisation, extraction, and resuspension.

Homogenisation

The first step is to break open the cells and suspend their contents in an extractable form.

  1. If your sample is cells in liquid (e.g. blood, spit), pelletise the sample by centrifuging. Discard the liquid.
  2. Suspend in 100ul homogenisation buffer, comprising 10mm edta, 60mm nacl, and 10mm tris (lowered to a pH of 7.5 using HCl)
  3. Add equal volume solubilisation buffer, comprising 2% SDS, 30mm EDTA, 200mm tris (lowered to a pH of 9 using HCl)
  4. Add 5ul/ml proteinase-k (i.e. 10 ul)
  5. Incubate at 37C for 1 hour

Extraction

  1. Add equal volume phenol-chloroform (i.e. 200ul)
  2. Emulsify by shaking (gently) or upending.
  3. Centrifuge at 13000 RPM for 10 minutes.
  4. 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

  1. Add 0.1 volumes sodium acetate (i.e. for 100ul of supernatant, add 10ul sodium acetate)
  2. Add 0.7 volumes IPA.
  3. Incubate at room temperature for 10-15 minutes
  4. Spin down at 13000 RPM for 10-15 minutes.
  5. Decant the IPA leaving only a glassy / clear pellet at the bottom. (IPA-precipitated pellets are glassy / clear; ethanol-precipitated pellets are white)
  6. Let the pellet dry completely so that no IPA remains, perhaps by incubating at 37C.
  7. Resuspend to 100ul with TE buffer, nuclease-free water, or R0 water.