Mar 06, 2019

Public workspaceE. coli K12 DNA Extraction

 Forked from E. coli K12 DNA Extraction
DOI
dx.doi.org/10.17504/protocols.io.yujfwun
  • 1University of Arizona
  • 481b Laboratory
Kenneth Schackart
Icon indicating open access to content
QR code linking to this content
DOI: dx.doi.org/10.17504/protocols.io.yujfwun
Protocol Citation: Kenneth Schackart, Kattika Kaarj 2019. E. coli K12 DNA Extraction. protocols.io https://dx.doi.org/10.17504/protocols.io.yujfwun
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 06, 2019
Last Modified: March 06, 2019
Protocol Integer ID: 21099
Abstract
How to extract DNA from E. coli K12 using Wizard® Genomic DNA Purification Kit by Promega®.

I do not claim any credit for the development of this protocol. It has been adapted from the protocol detailed in: Download Wizard Genomic DNA Purification.pdfWizard Genomic DNA Purification.pdf .

Guidelines
Lab coat and gloves must be worn at all times.
Materials
MATERIALS
ReagentWizard(R) Genomic DNA Purification KitPromegaCatalog #A1620
Reagents in kit:
  • Nuclei Lysis Solution
  • RNAse Solution
  • Protein Precipitation Solution
  • DNA Rehydration Solution

Additional Reagents:
  • Isopropanol
  • 70% Ethanol

Additional Materials:
  • 2 mL centrifuge tubes
  • Pipettes and tips
  • Heating block
  • Centrifuge
Before start
Spray work area with 70% EtOH solution.
Culture bacteria
Culture bacteria
Culture E. coli K12 in BHI broth overnight.
Amount2 mg lyophilized E. coli K12 in Amount10 mL BHI broth.


Pellet the cells
Pellet the cells
Add Amount1 mL cell suspension to 2 mL microcentrifuge tube.

Label centrifuge tube with your group number.
Centrifuge at 13,000-16,000 × g for Duration00:02:00 .

Remove supernatant.
Lyse nuclei
Lyse nuclei
Add Amount600 µL of Nuclei Lysis Solution.
Note
Nuclei Lysis Solution is marked "NL"


Gently pipet until the cells are resuspended.
Incubate atTemperature80 °C on heating block for Duration00:05:00 to lyse the cells.


Cool to room temperature.
Degrade RNA
Degrade RNA
Add Amount3 µL RNase Solution to the cell lysate.
Note
RNAse solution is marked "R"


Invert 2-5 times to mix.
Incubate at Temperature37 °C for Duration00:15:00 to Duration01:00:00 .



Cool to room temperature.
Precipitate proteins
Precipitate proteins
Add Amount200 µL of Protein Precipitation Solution to the RNase-treated cell lysate.

Note
Protein Precipitation solution is marked "P"

Vortex vigorously at high speed for Duration00:00:20 .

Incubate on ice for Duration00:05:00

Centrifuge at 13,000-16,000 × g for Duration00:03:00 .
Harvest DNA
Harvest DNA
Transfer the supernatant containing the DNA to a clean 1.5 mL microcentrifuge tube containing Amount600 µL isopropanol.
Note
Some supernatant may remain in the original tube conatining the protein pellet. Leave this residual to avoid contaminating the DNA solution with the precipitated protein.

Note
Isopropanol is marked "IPA"


Label centrifuge tube with your group number.
Gently mix by inversion until the thread-like strands of DNA form a visible mass.
Wash and dry DNA
Wash and dry DNA
Centrifuge at 13,000-16,000 × g for Duration00:02:00 .
Carefully pour off the supernatant and drain the tube on clean absorbent paper.
Add Amount600 µL of 70% ethanol and gently invert the tube several times to wash the DNA pellet.

Note
70% ethanol solution is marked "Et"

Centrifuge at 13,000-16,000 × g for Duration00:02:00 .
Carefully pour off the ethanol.
Drain the tube on clean absorbent paper and allow to air-dry for 10-15 minutes.
Rehydrate DNA
Rehydrate DNA
Add Amount100 µL of DNA rehydration solution to the tube.

Note
DNA Rehydration solution is marked "DR"

Rehydrate by incubating the solution overnight at room temperature or Temperature4 °C .

Store DNA at Temperature2 °C to Temperature8 °C .