Dec 13, 2021

Public workspaceDNA Extraction from Bacteriophages

This protocol is a draft, published without a DOI.
  • 1Copenhagen University
  • FOOD Micro UCPH
Frej Larsen
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Protocol Citation: Frej Larsen 2021. DNA Extraction from Bacteriophages. protocols.io https://protocols.io/view/dna-extraction-from-bacteriophages-b2tmqek6
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: December 13, 2021
Last Modified: December 13, 2021
Protocol Integer ID: 55885
Abstract
DNA/RNA extraction is performed using the QIAamp® Viral RNA Mini kit from Qiagen without the addition of carrier RNA to the AVL buffer and an additional nuclease step prior to extraction. The nuclease step is introduced to degrade any DNA/RNA that may be in the sample after the virome extraction protocol (dx.doi.org/10.17504/protocols.io.b2qaqdse) has been performed. Please note that even though the kit is a viral RNA extraction kit, viral DNA will also be extracted.
Guidelines
Before beginning this protocol, ensure that wash buffers have been properly diluted with ethanol and that the centrifuge is available and not chilled as low temperatures may impede ethanol evaporation.

Note that, unlike when using the kit directly, carrier RNA should not be added to the AVL buffer. The Pierce Universal nuclease should be diluted 100 times (ie. by mixing 1 uL nuclease with 99 uL SM buffer/sterile water)

Wear gloves when performing this protocol.
For each sample, pipet Amount1 µL 100x diluted Pierce Universal Nuclease (2.5units/uL) to an empty 1.5 mL tube.

Transfer Amount140 µL of each sample from the outer chamber of the CentrisArt filter tube to the tube and mix by pipetting

Incubate for at least Duration00:03:00 at room temperature.

3m
Add Amount540 µL AVL buffer to inactivate nucleases and lyse phage heads. Mix by pipetting or pulse vortexing.

Incubate Duration00:10:00 at room temperature

10m
Change gloves.
Briefly centrifuge samples with a microcentrifuge.
Add Amount560 µL absolute ethanol. Mix thoroughly by pulse vortexing.

Briefly centrifuge samples with a microcentrifuge
Transfer Amount630 µL of the sample to a spin column

Centrifuge Centrifigation6000 x g, 21°C, 00:01:00 or until all liquid has passed through the filter.

1m
Change the collection tube and Go togo to step #10 until all of the sample has passed through the filter.

Add Amount500 µL AW1 wash buffer to each spin column.

Centrifuge Centrifigation6000 x g, 21°C, 00:01:00 or until all liquid has passed through the filter
1m
Add Amount500 µL AW2 wash buffer to each spin column.

Centrifuge Centrifigation20000 x g, 21°C, 00:03:00 or until all liquid has passed through the filter.
3m
Change the collection tube, then centrifuge Centrifigation20000 x g, 21°C, 00:01:00 to dry the membrane.
1m
Place the spin column in a new RNAse-free 1.5 mL tube.
Add Amount30 µL AVE elution buffer directly onto the filter membrane in the spin column.

Incubate at room temperature for at least Duration00:01:00 .

1m
Centrifuge Centrifigation6000 x g, 21°C, 00:01:00
1m
Discard the spin column and store eluate at Temperature-80 °C