Nov 25, 2020
OG1RF_genomic_DNA
This protocol is a draft, published without a DOI.
- 1In-house protocol
Protocol Citation: Elizabeth Fozo 2020. OG1RF_genomic_DNA. protocols.io https://protocols.io/view/og1rf-genomic-dna-bp4tmqwn
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: November 25, 2020
Last Modified: November 25, 2020
Protocol Integer ID: 44915
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Abstract
OG1RF genomic DNA isolation
Steps
Steps
Generate an overnight culture in 10 mL of BHI
Spin down culture in morning for 10 min at 3500 RPM and pour off supernatant.
Make 50 mM EDTA (500 µL 0.5M EDTA stock in 4.5 mL water).
Add 960 µL of 50 mM EDTA to OG1RF pellet, then aliquot 480 µL of 50 mM EDTA/cells to a 1.5 mL Eppendorf tube.
Add 120 µL of 10 mg/mL lysozyme
Incubate at 37°C for 60 minutes
Spin at 13000 RPM for 2 minutes and pour off supernatant.
Add 600 µL nuclei lysis solution
Incubate at 80°C for 5 min then cool to room temperature
Add 3 µL of RNAse solution
Incubate at 37°C for 60 minutes
Add 200 µL of protein precipitation solution. Vortex vigorously for 20 seconds
Put on ice for 5 minutes
Spin at 13000 RPM for 3 minutes
Transfer supernatant to a clean Eppendorf tube containing 600 µL of isopropanol
Gently invert
Spin at 13000 RPM for 2 minutes and pour off supernatant. Be careful to not disturb pellet.
Add 600 µL of 70% ethanol to wash DNA pellet. Stream ethanol directly onto pellet without touching the pipette tip to the pellet.
Spin at 13000 RPM for 2 minutes and pour off supernatant carefully.
Invert and air dry
Add 100 µL of DNA hydration solution
Incubate at 65°C for 1 hour or leave at 4°C overnight