Mar 26, 2024
Phenol-chloroform DNA extraction from Sporosarcina pasteurii
- Michael S. Carter1,
- Matthew J. Tuttle1,2,
- Maneesh K. Gupta1
- 1Materials and Manufacturing Directorate, Air Force Research Laboratory, Wright-Patterson Air Force Base, Dayton, Ohio, USA;
- 2Biological and Nanoscale Technologies Division, UES Inc., a BlueHalo Company, Dayton, Ohio, USA
Protocol Citation: Michael S. Carter, Matthew J. Tuttle, Maneesh K. Gupta 2024. Phenol-chloroform DNA extraction from Sporosarcina pasteurii. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg324eev25/v1
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: March 25, 2024
Last Modified: March 26, 2024
Protocol Integer ID: 97344
Keywords: DNA extraction, Sporosarcina pasteurii
Abstract
This protocol is for extraction of genomic DNA from Sporosarcina pasteurii. It is based on a standard phenol-chloroform DNA extraction method.
Attachments
Guidelines
All steps should be performed in a chemical fume hood.
Materials
• Brain-heart infusion (BHI) broth supplemented with 330 mM urea
• Resuspension buffer (50 mM Tris pH 8.0, 10% sucrose)
• Lysis buffer (Tris pH 8.0, 10 mg/mL lysozyme)
• 10% sodium dodecyl sulfate (SDS)
• 100 mg/mL RNase A
• 50 mg/mL proteinase K
• 3.0 M sodium acetate, pH 5.5
• 100% ethanol
• TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0)
• Phenol:chloroform:isoamyl alcohol (25:24:1, saturated with 10mM Tris, pH 8.0, 1mM EDTA)
• Chloroform
• 70% ethanol
DNA extraction
DNA extraction
Grow Sporosarcina pasteurii cells in 150 mL of BHI/330 mM urea at 30°C with 200 rpm shaking to an OD600 of 3.5.
Concentrate cells by centrifugation at 15,000 x g for 10 min and pour off the supernatant.
Resuspend cells in 10 mL of Resuspension Buffer (50 mM Tris pH 8.0, 10% sucrose).
Add 2.5 mL Lysis Buffer (Tris pH 8.0, 10 mg/mL lysozyme), 1.5 mL 10% SDS, 5 µL 100 mg/mL RNase A, and 25 µL 50 mg/mL proteinase K to lyse cells and stabilize DNA.
Incubate for 1 h at 37°C.
Add 1.3 mL of 3.0 M sodium acetate (pH 5.5) and 30 mL of 100% ethanol to separate DNA from other biomacromolecules by precipitation.
Spool DNA onto a glass hook and transfer to 20 mL of TE buffer.
Dissolve DNA in TE buffer for 1 h at 37°C.
Add a 5 mL aliquot of DNA to 7 mL of phenol:chloroform:isoamyl alcohol (25:24:1, Saturated with 10mM Tris, pH 8.0, 1mM EDTA) and mix by inversion.
Separate phases by centrifugation at 10,000 x g for 10 min.
Pipette off the aqueous phase and add to 5 mL of chloroform. Mix by vortexing.
Separate phases by centrifugation at 10,000 x g for 10 min.
Pipette off the aqueous phase and add to 30 mL of 100 % ethanol. Mix by inversion.
Collect precipitated DNA by centrifugation at 10,000 x g for 10 min.
Wash pelleted DNA twice by adding 5 mL of 70% ethanol, mixing by inversion, centrifuging at 10,000 x g for 5 min, and removing the supernatant.
Allow the pellet to dry.
Dissolve the final pellet in 500 µL of TE buffer at 37°C for 1 h.
Protocol references
Distribution A. Approved for public release: distribution unlimited. AFRL-2024-1482.