PhenolChloroformExtraction < Lab

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---+ 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) %RED% pH 8.05 %ENDCOLOR%
      * 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)

<b>Lysis Buffer: </b>
| *Reagent* | *amount for x10* |
| TE buffer | 5.85 mL |
| RNaseA | 20  µL |
| Lysozyme | 20 mg |
* Add enzymes to TE buffer

<b>TE Buffer: </b>
| *Reagent* | *Stock Conc.* | *to 50 mL* |
| Tris-HCl pH 8.0 | 1 M | 500 µL |
| EDTA pH 8.0 | 0.5 M | 100  µL |
| H<sub>2</sub>O | - | 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.

<b>3.15 M NaCl: </b>
| *Reagent* | *500 mL* |
| NaCl | 92 g |
| H<sub>2</sub>O | to 500 mL |


---+ Equipment

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

---+ Procedure

---+++ =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
   1 incubate 30 min @ 37ºC, gentle rocking
   1 add 30 µL 10% SDS
   1 incubate 30 min @37ºC
---+++ =Add salt=
   1 add 180 µL 3.15 M NaCl and vortex
   1 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
   1 microcentrifuge 5 min @ max
   1 remove 550 µL supernatant, put in new tube
      * place into new microcentrifuge tube
   1 add 550 µL phenol:chloro:iso and vortex
   1 microcentrifuge 5 min @ max 
   1 remove 300 uL supernatant, put in new tube
---+++ =DNA precipitation=
   1 add 180 µL (0.6 * 300 µL) 100% isopropyl and vortex
   1 incubate 15 min RT
   1 microcentrifuge 5 min @ max
   1 discard supernatant 
   1 add 1 mL 70% EtOH
   1 microcentrifuge 5 min
   1 discard EtOH supernatant
   1 dry 10 min, vacuum spin
   1 add 85 µL TE 
   1 incubate overnight @ 42ºC
      * alternatively incubate 10 min 65ºC
   1 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
Barrick Lab > ProtocolList > PhenolChloroformExtraction
Topic revision: r2 - 2022-07-20 - 16:42:59 - Main.MattMcGuffie