Phenol/chloroform extraction

* Extracting genomic bacterial DNA from pellet with phenol/chloroform with a combined EtOH precipitation step for salt/SDS/small nucleic acid removal.*

Materials

  • Lysis buffer (see below)
  • 10% SDS solution (w/v)
  • 3.15 M NaCL solution (see below)
  • Chloroform
  • Isoamyl alcohol
  • Phenol:Chloroform:Isoamyl Alcohol (25:24:1, v/v) pH 8.05
    • pH is very important
    • solution should be < 1 year old and not oxidized (i.e. clear color)
  • 100% isopropyl alchohol
  • 70% EtOH
  • TE buffer
    • commercial or prepare your own (see below)

Lysis Buffer:

Reagent amount for x5
TE buffer 5.85 mL
RNaseA 20 無
Lysozyme 20 mg
* Add enzymes to TE buffer

TE Buffer:

Reagent Stock Conc. to 50 mL
Tris-HCl pH 8.0 1 M 500 無
EDTA pH 8.0 0.5 M 100 無
H2O - 49.4 mL
* For a 1 M Tris-HCl pH 8.0 solution, dissolve 121.1 g of Tris base in 800 ml of H2O. Adjust the pH to the desired value by adding concentrated HCl.

3.15 M NaCl:

Reagent 500 mL
NaCl 92 g
H2O to 500 mL

Equipment

  • high speed, fixed-angle rotor microcentrifuge
  • 37慢 incubator
  • 65慢 water bath
  • 42慢 water bath

Procedure

Lysis

  1. resuspend bacterial pellet in 570 無 Lysis Buffer
    • typically I use 1 mL o/n culture, pellet frozen at -80 C for at least an hour
  2. incubate 30 min @ 37慢, gentle rocking
  3. add 30 無 10% SDS
  4. incubate 30 min @37

Add salt

  1. add 180 無 3.15 M NaCl and vortex
  2. incubate 10 min @ 65慢
    • uncertain if incubation step is necessary

Extraction

  1. add 700 無 24:1 chloroform:isoamyl alcohol solution and vortex
    • NOTE: this is not phenol:chloroform:isoamyl alcohol
  2. microcentrifuge 5 min @ max
  3. remove 550 無 supernatant, put in new tube
    • place into new microcentrifuge tube
  4. add 550 無 phenol:chloro:iso and vortex
  5. microcentrifuge 5 min @ max
  6. remove 300 uL supernatant, put in new tube

DNA precipitation

  1. add 180 無 (0.6 * 300 無) 100% isopropyl and vortex
  2. incubate 15 min RT
  3. microcentrifuge 5 min @ max
  4. discard supernatant
  5. add 1 mL 70% EtOH
  6. microcentrifuge 5 min
  7. discard EtOH supernatant
  8. dry 10 min, vacuum spin
  9. add 85 無 TE
  10. incubate overnight @ 42慢
    • alternatively incubate 10 min 65慢
  11. vortex tubes after incubation

NOTES

  • Proteinase K is omitted from this protocol. Unfolded proteins efficiently separate into the chloroform phases, while short peptides may be retained in the aqueous phase
  • The NaCl is for EtOH precipitation later, though also aids in protein unfolding
  • The initial chloroform extraction (Extraction step 1) greatly improved the efficiency and purity of DNA in tests
  • Be careful to only remove the aqueous phase during steps involving chloroform. Try not to disturb the interphase
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Contributors to this topic Edit topic MattMcGuffie
Topic revision: r1 - 2021-11-03 - 20:49:44 - Main.MattMcGuffie
 
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