DNA Extraction from Bacteriophages - 96 well format
Frej Larsen
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.
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.
Steps
Pipet 1µL 100x diluted Pierce Universal Nuclease (2.5units/uL) to each well of a Qiagen S-block
Transfer 140µL of each sample from the outer chamber of the CentrisArt filter tube to the S-block. Mix by pipetting
Incubate for 0h 5m 0s at room temperature
Add 540µL AVL buffer to inactivate nucleases and lyse phage heads. Mix by pipetting
Incubate 0h 10m 0s at room temperature
Add 560µL absolute ethanol. Mix thoroughly by pipetting
Place a QIAamp 96 plate on a new S-block
Transfer 630µL 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 5500x g,21°C or until all liquid has passed through the filter
Repeat steps 8 and 9 until all of the sample has passed through the filter
Replace the bottom S-block and add 500µL 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 5500x g,21°C or until all liquid has passed through the filter
Remove the seal, add 500µL AW2 wash buffer to each well, then reseal the plate.
Load the QIAamp 96 plate on the S-block into a rotor bucket. Centrifuge 5500x g,21°C or until all liquid has passed through the filter
Place the QIAamp 96 plate on a new S-block and centrifuge 5500x g,21°C to dry the membrane
Discard the S-block and place the QIAamp 96 plate on an A&A receiving plate. Add 60µL AVE elution buffer directly onto the filter membrane
Seal the plate with an AirPore Tape sheet, then incubate at room temperature for 0h 10m 0s
Load the QIAamp 96 plate on the receiving plate into a rotor bucket. Centrifuge 5500x g,21°C
Discard the QIAamp 96 plate, seal the receiving plate for storage and store at -80°C
