Modified Phenol Chloroform Genomic DNA Extraction Protocol from the Christner Lab (University of Florida)
Shelby J Barnes, Noelle Bryan, Brent Christner, Cameron Thrash
Disclaimer
Lead author S. Barnes did not create protocol but is submitting with permission of author N. Bryan.
Bryan, N.C., Christner, B.C., Guzik, T.G. et al. Abundance and survival of microbial aerosols in the troposphere and stratosphere. ISME J 13, 2789–2799 (2019). https://doi.org/10.1038/s41396-019-0474-0
Christner Lab protocol reference:
Sambrook J, Russell DW, editors. Molecular cloning: a laboratory manual. Cold Spring Harbor Cold Spring Harbor, USA: Laboratory Press; 2001.
Abstract
Modified Phenol Chloroform genomic DNA Extraction Protocol from the Christner Lab (University of Florida)
Steps
Lysis
Start with filtered cells OR spin to get pellet from liquid culture.
Pipette off supernatant, if any.
Add 380 uL 1x TE Buffer + 20 uL Lysozyme to sample.
Incubate for 1 h at 37˚C.
Add 60 uL SDS + 30 uL Proteinase K + 110 uL 1x TE Buffer (total volume = 600 uL) to sample.
Incubate for 2 h at 37˚C.
Vortex tube thoroughly.
Incubate at 37˚C for 1 h , or until samples are clear.
Incubate at 65˚C for 30 min to inactivate Proteinase K.
Stop here and store at -20˚C or move to extraction.
Phenol Chloroform Extraction
Add equal volume (600 uL) of Phenol Chloroform Isoamyl alcohol (P/C/I) to sample.
Invert samples thoroughly before centrifuge step.
Spin at 10,640 rcf for 10 min.
White interface should be visible.
Transfer aqueous phase (top layer) to new tube.
Transfer approx. 500 - 600 uL in 100 uL increments.
Do not disturb interface.
Add equal volume of Chloroform Isoamyl alcohol (C/I) to sample.
Approx. 500 uL
Invert sample thoroughly before centrifuge step.
Spin at 10,640 rcf for 10 min.
White interface should be visible.
Transfer aqueous phase (top layer) to new tube.
Do not disturb interface.
Approx. 300 - 500 uL
Store at -20˚C or move to ethanol precipitation.
Ethanol Precipitation
Add 2 volumes of ice cold 100% Ethanol to sample.
E.g. Have 50 uL of DNA solution, add 100 uL Ethanol.
Add 0.1 volumes of 3M Sodium Acetate pH 5.2 to sample.
E.g. Have 50 uL DNA solution, add 5 uL Sodium Acetate.
Incubate at -20˚C for at least 1 hr.
Cold incubation for a longer period of time (e.g. over night) may lead to higher yield.
Spin at 13,000 rcf for 30 min at 4˚C .
Should see small white pellet.
If tube is placed hinge-side outward, the pellet should be found below the hinge and almost to the bottom.
Pipette off supernatant.
Gently add 150 uL of 70% ethanol to remove salts.
Do not dislodge pellet.
Keeping the samples on ice or in cold tube racks will help pellet stay in place.
Spin at 13,000 rcf for 10 min at 4˚C .
Pipette off supernatant.
Spin at 13,000 rcf for 1 min to bring down any excess ethanol.
Pipette off any remaining liquid.
Dry pellet for 5 min MAX.
Any longer makes resuspending difficult.
Resuspend pellet in 50 uL Molecular Bio water.
Use 1- 3 uL of DNA to quantify using Qubit HS dsDNA assay kit.
Store at -20˚C .
