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.*

• 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 x10 TE buffer 5.85 mL RNaseA 20 µL Lysozyme 20 mg * Add enzymes to TE buffer TE Buffer: Reagent Stock Conc. to 50 mL Tris-HCl pH 8.0 1 M 500 µL EDTA pH 8.0 0.5 M 100 µL 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

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

Lysis

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

Add salt

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

Extraction

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

DNA precipitation

1. add 180 µL (0.6 * 300 µL) 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 µL TE
10. incubate overnight @ 42ºC
    • alternatively incubate 10 min 65ºC
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