Feb 24, 2022
Version 2
Modified Phenol Chloroform Genomic DNA Extraction Protocol from the Christner Lab (University of Florida) V.2
- Shelby J Barnes1,
- Noelle Bryan2,
- Brent Christner3,
- Cameron Thrash1
- 1University of Southern California;
- 2Louisiana State University;
- 3University of Florida
Protocol Citation: Shelby J Barnes, Noelle Bryan, Brent Christner, Cameron Thrash 2022. Modified Phenol Chloroform Genomic DNA Extraction Protocol from the Christner Lab (University of Florida). protocols.io https://dx.doi.org/10.17504/protocols.io.b5iiq4ceVersion created by Shelby J Barnes
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it’s working
Created: February 23, 2022
Last Modified: February 24, 2022
Protocol Integer ID: 58666
Keywords: Phenol Chloroform Extraction , Genomic DNA Extraction
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 J13,2789–2799 (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)
Materials
Lysis Step:
10% SDS
TE Buffer (1x) - diluted with Molecular Bio Water
Proteinase K (20 mg/mL) - in Molecular Bio Water
Lysozyme (10 mg/mL) - in Molecular Bio Water
Phenol Chloroform Extraction Step:
Phenol Chloroform Isoamyl alcohol (25:24:1)
Chloroform Isoamyl alcohol (24:1)
3 - 2 mL microcentrifuge tubes per sample
Ethanol Precipitation Step:
Ice cold 100% Ethanol
3M Sodium Acetate pH 5.2
70% Ethanol - diluted with Molecular Bio Water
Molecular Bio Water
Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Filter with 0.2 um PES filter before use:
10% SDS
1x TE Buffer
100% Ethanol
70% Ethanol
All Molecular Bio Water used as reagent or diluent
Safety warnings
Take precaution when working with Phenol Chloroform Isoamyl Alcohol and Chloroform Isoamyl Alcohol.
Use eye protection and work in highly ventilated space like fume hood.
Phenol Chloroform Isoamyl Alcohol
H301 Toxic if swallowed. H312 + H332 Harmful in contact with skin or if inhaled. H314 Causes severe skin burns and eye damage. H336 May cause drowsiness or dizziness. H341 Suspected of causing genetic defects. H351 Suspected of causing cancer. H361 Suspected of damaging fertility or the unborn child. H372 Causes damage to organs (Liver, Kidney) through prolonged or repeated exposure if swallowed. H373 May cause damage to organs (Nervous system, Kidney, Liver, Skin) through prolonged or repeated exposure.
Chloroform Isoamyl Alcohol
H227 Combustible liquid. H302 Harmful if swallowed. H315 Causes skin irritation. H318 Causes serious eye damage. H331 Toxic if inhaled. H336 May cause drowsiness or dizziness. H351 Suspected of causing cancer. H361 Suspected of damaging fertility or the unborn child. H372 Causes damage to organs (Liver, Kidney) through prolonged or repeated exposure if swallowed.
Lysis
Lysis
5h
5h
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
Phenol Chloroform Extraction
1h
1h
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
Ethanol Precipitation
2h
2h
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 .