<noautolink> ---+ 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
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Topic revision: r2 - 2022-07-20 - MattMcGuffie