MODIFIED DNA ISOLATION PROTOCOL | Anisa M.

Modified extraction protocol for DNA

isolation using Quick-gDNA Miniprep kit
from bacterial samples.
Disclaimer: The DNA isolation protocol provided here is a modified version of two separate
protocols from different manufacturers, the Qiagen DNeasy Blood & Tissue Kit and the
Zymo Quick-gDNA Miniprep Plus Kit. This protocol is not a validated lab protocol and has
not been tested or approved by either manufacturer. The contents of the Qiagen and Zymo
kits have been used in this new protocol, but modifications have been made to suit the
specific needs of the user. It is recommended that users perform appropriate optimization and
validation steps to ensure the suitability and reliability of the protocol for their specific
applications.

The protocol:
1. Harvest the bacterial cells by centrifuging the culture at high speed (e.g. 10,000-15,000 x
g) for several minutes. Discard the supernatant and carefully resuspend the bacterial pellet in
a small volume of phosphate-buffered saline (PBS) or other appropriate buffer.
2. Add the resuspended bacterial cells to a microcentrifuge tube containing Genomic lysis
buffer from the Quick-gDNA Miniprep kit. Use a volume of lysis buffer appropriate for the
volume of bacterial suspension (e.g. 200-500 µL of Genomic lysis buffer for 1 mL of
bacterial culture).
3. Incubate the bacterial cells and Genomic lysis buffer at 37°C for 30-60 minutes to allow
for cell lysis.
4. Add 20μL Proteinase K Solution the lysate and incubate for an additional 10-20 minutes at
55°C to digest proteins and RNA.
5. Add ethanol to the lysate to facilitate DNA binding to the silica membrane in the Zymo-
spin column in a Collection Tube of the kit.
6. Transfer the lysate to the Zymo-spin column, and centrifuge at high speed to separate the
DNA from other cellular components. Discard the Collection Tube with the flow through.
7. Transfer the Zymo-spin column to a new Collection Tube. Add 200 µL of DNA Pre-
Wash Buffer to the spin column and centrifuge at 10,000 x g for one minute. This wash
buffer provided in the kit will remove residual contaminants.
8. Add 500 µL of g-DNA Wash Buffer to the spin column. Centrifuge at 10,000 x g for one
minute.

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 MODIFIED DNA ISOLATION PROTOCOL | Anisa M.

9. Transfer the spin column to a clean microcentrifuge tube. Add ≥50μL of DNA elution
buffer to the spin column. Incubate for 2-5 minutes at room temperature and then centrifuge
at top speed for 30 seconds to elute the DNA.
Unfortunately, we currently do not have a complete kit for bacterial DNA extraction in our
section. It's important to note that this protocol may not be as efficient as a bacterial DNA
extraction kit specifically designed for this purpose, and the quality and quantity of DNA
obtained may be lower. Additionally, the lysis buffer provided in the Quick-gDNA Miniprep
kit may not be as effective at lysing bacterial cells as a specialized bacterial lysis buffer.
However, this protocol may still be useful for obtaining DNA from bacterial samples when
other options are not available, especially following optimization.

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