Nov 25, 2021

Public workspaceDNA Extraction from Bacteriophages - 96 well format

DOI
https://dx.doi.org/10.17504/protocols.io.bzkup4ww
  • Frej Larsen1
  • 1Copenhagen University
  • FOOD Micro UCPH
Frej Larsen
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DOI: https://dx.doi.org/10.17504/protocols.io.bzkup4ww
Protocol Citation: Frej Larsen 2021. DNA Extraction from Bacteriophages - 96 well format. protocols.io https://dx.doi.org/10.17504/protocols.io.bzkup4ww
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: October 29, 2021
Last Modified: November 25, 2021
Protocol Integer ID: 54644
Keywords: well format extraction of viral dna, dna extraction from bacteriophage, qiagen kit qiaamp viral rna mini kit, dna extraction, viral dna, rna from culture lysate, well format extraction, bacteriophage, samples free from bacteria, dna, rna, pierce universal nuclease
Abstract
Extraction of viral DNA/RNA from culture lysate or filtered samples free from bacteria. The protocol is based on the Qiagen kit QIAamp Viral RNA Mini kit for 96 well plates and uses consumables from this kit as well as the Pierce Universal Nuclease.

Troubleshooting
Before start
Before beginning this protocol, ensure that wash buffers have been properly diluted with ethanol and that the centrifuge is available and not chilled at low temperatures may impede ethanol evaporation.
Pipet Amount1 µL 100x diluted Pierce Universal Nuclease (2.5units/uL) to each well of a Qiagen S-block

Transfer Amount140 µL of each sample from the outer chamber of the CentrisArt filter tube to the S-block. Mix by pipetting

Incubate for Duration00:05:00 at room temperature

5m
Add Amount540 µL AVL buffer to inactivate nucleases and lyse phage heads. Mix by pipetting

Incubate Duration00:10:00 at room temperature

10m
Add Amount560 µL absolute ethanol. Mix thoroughly by pipetting

Place a QIAamp 96 plate on a new S-block
Transfer Amount630 µL to the QIAamp 96 plate, then seal it with an AirPore tape sheet

Load the QIAamp 96 plate on the S-block into a rotor bucket. Centrifuge Centrifigation5500 x g, 21°C, 00:04:00 or until all liquid has passed through the filter

4m
Repeat steps 8 and 9 until all of the sample has passed through the filter

Replace the bottom S-block and add Amount500 µL AW1 wash buffer to each well, then seal the QIAamp 96 plate

Load the QIAamp 96 plate on the S-block into a rotor bucket. Centrifuge Centrifigation5500 x g, 21°C, 00:05:00 or until all liquid has passed through the filter
5m
Remove the seal, add Amount500 µL AW2 wash buffer to each well, then reseal the plate.

Load the QIAamp 96 plate on the S-block into a rotor bucket. Centrifuge Centrifigation5500 x g, 21°C, 00:05:00 or until all liquid has passed through the filter
5m
Place the QIAamp 96 plate on a new S-block and centrifuge Centrifigation5500 x g, 21°C, 00:10:00 to dry the membrane
10m
Discard the S-block and place the QIAamp 96 plate on an A&A receiving plate. Add Amount60 µL AVE elution buffer directly onto the filter membrane

Seal the plate with an AirPore Tape sheet, then incubate at room temperature for Duration00:10:00

10m
Load the QIAamp 96 plate on the receiving plate into a rotor bucket. Centrifuge Centrifigation5500 x g, 21°C, 00:04:00
4m
Discard the QIAamp 96 plate, seal the receiving plate for storage and store at Temperature-80 °C