Phenol Chloroform extraction of DNA
The phenol-chloroform method separates nucleic
acids (DNA and RNA) from proteins and lipids based on their
solubility in aqueous vs. organic phases.
·
Phenol denatures proteins.
·
Chloroform helps in sharper phase separation.
·
Centrifugation separates the mixture into:
o Aqueous phase (top) → contains DNA
o Interphase → proteins
o Organic phase (bottom) → phenol, chloroform, lipids
·
Sodium acetate neutralizes the
negative charges on the DNA backbone (phosphates), allowing strands to come
together.
·
Ethanol reduces the dielectric
constant of the solution, which promotes aggregation and precipitation of the
neutralized DNA
It does
not differentiate DNA sources (host vs. viral) because both host and viral
DNA are chemically similar and partition into the aqueous phase.
Reagents and
Equipment Required
·
Lysis buffer
- 100 mM Tris-HCl (pH 8.0): 1 mL of 1 M
stock
- 50 mM EDTA (pH 8.0): 1 mL of 0.5 M stock
- 100 mM NaCl: 0.2 mL of 5 M stock
- 1% SDS: 1 mL of 10% stock
- Add nuclease-free water to 10 mL
·
Proteinase K (20 mg/mL)
- Add proteinase K into lysis buffer so
that final concentration will be 100-200 ug/mL.
- For 20 mg/mL solution, it is 1 uL into
800 uL solution.
- Note, Prepare this solution when you are
about to extract DNA.
·
Phenol: Chloroform: Isoamyl
alcohol (25:24:1)
- 25 mL saturated phenol (pH 8.0)
- 24 mL chloroform
- 1 mL isoamyl alcohol
- Mix well and store at 4°C in dark bottle
·
Sodium acetate (3M, pH 5.2) or
NaCl
·
Ethanol (100% and 70%, cold)
·
TE buffer or nuclease-free
water
·
Microcentrifuge tubes
·
Vortex
·
Microcentrifuge
🧬 Procedure
1. Cell Lysis
- Add sample (cells/tissue) into a
microcentrifuge tube. (200uL)
- Add appropriate volume of lysis
buffer + Proteinase K. (200uL)
- Incubate at 55°C for 15-30
minutes (until lysed).
2.
Phenol:Chloroform Extraction
- Add equal volume of
phenol:chloroform:isoamyl alcohol (25:24:1). (400uL)
- Vortex vigorously for 15–30
seconds.
- Centrifuge at 12,000–14,000 rpm
for 10 minutes at room temperature.
- Carefully transfer the top
aqueous layer (contains DNA) into a new tube.
(300 uL approx..)
- Note: Do not be greedy while
transferring top layer. This may result in protein contamination.
4. DNA
Precipitation
- Add 0.1 volume of 3M sodium
acetate (pH 5.2) or NaCl. (35uL)
- Add 2 volumes of cold 100%
ethanol. (770 uL)
- Mix gently and incubate at –20°C
for 30 minutes.
- Centrifuge at 12,000 rpm for
10–15 minutes to pellet DNA.
5. Wash DNA
Pellet
- Remove supernatant.
- Wash pellet with 500 µL of 70%
ethanol.
- Centrifuge briefly (5 minutes),
discard ethanol.
- Air-dry or vacuum-dry the DNA pellet.
6. Resuspend
DNA
- Dissolve DNA in TE buffer
or nuclease-free water (20–50 µL).
- Store at 4°C short-term, or
–20°C long-term.
Use Nanodrop:
A260/A280 ratio ~1.8 indicates pure DNA.