- Materials
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- DNAseI treated rAAV lysate samples
- Consumables
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- PureLink™ Viral RNA/DNA Mini Kit (Thermo Fisher, 12280050)
- Qubit 1x dsDNA HS Assay Kit (ThermoFisher, Q33230)
- Qubit™ ssDNA Assay Kit (ThermoFisher, Q10212)
- Ethanol, 100% (e.g. Fisher, 16606002)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 2 ml Eppendorf DNA LoBind tubes
- 1.5 ml Eppendorf DNA LoBind tubes
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- Equipment
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- Microfuge
- Vortex mixer
- Thermal cycler
- Eppendorf 5424 centrifuge (or equivalent)
- P1000 pipette and tips
- P100 pipette and tips
- P200 pipette and tips
- P20 pipette and tips
- P10 pipette and tips
- Qubit fluorometer (or equivalent for QC check)
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This extraction step is performed using the PureLink™ Viral RNA/DNA Mini Kit and includes steps from the user guide for completeness.
The PureLink™ Viral RNA/DNA Mini Kit user guide is available here.
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Add 60 ml of 96-100% ethanol to 15 ml of Wash Buffer (WII) and store at room temperature.
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Add 310 µl nuclease-free water directly to the tube of 310 µg lyophilised Carrier RNA to obtain 1 µg/µl Carrier RNA stock solution and mix thoroughly.
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Calculate the volume of Lysis Buffer/Carrier RNA mix required to process the desired number of samples simultaneously using the following formula:
N x 0.21 ml (volume of Lysis Buffer/reaction) = A ml
A ml x 28 µl/ml = B µlN = number of samples
A = calculated volume of Lysis Buffer
B = calculated volume of 1 µg/µl Carrier RNA stock solution to add to Lysis BufferWorked example for 6 samples:
6 x 0.21 ml = 1.26 ml
1.26 ml x 28 µl/ml = 35.28 µl1.26 ml of Lysis Buffer
35.28 µl of Carrier RNA stock solution -
Aliquot the Carrier RNA stock solution and take forward the required volume into the next step. Store any excess aliquots at -20°C and avoid repeated freezing and thawing.
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In a sterile 2 ml Eppendorf DNA LoBind tube, add the calculated volume of Carrier RNA stock solution to the calculated volume of Lysis Buffer and mix gently by pipetting.
For 6 samples, add 35.28 µl of Carrier RNA stock solution to 1.26 ml of Lysis Buffer.
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Store the buffer at 4°C until use.
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Add 25 µl Proteinase K into a fresh 1.5 ml Eppendorf DNA LoBind tube for each sample.
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Spin down the DNAseI treated rAAV samples.
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Transfer 200 µl of an rAAV lysate sample to a tube containing Proteinase K and repeat for each sample into separate tubes.
Note: Ensure the rAAV samples are at room temperature and not combined.
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Add 200 µl Lysis Buffer to each tube. Close the tube lids and mix by vortexing for 15 seconds.
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Incubate at 56°C for 15 minutes.
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Briefly centrifuge the tubes to remove any drops from the inside of the lids.
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Add 250 µl 96-100% ethanol to each tube to obtain a final concentration of 37% and mix by vortexing for 15 seconds.
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Incubate the tubes with ethanol for 5 minutes at room temperature and spin down.
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Spin down the tubes to remove any drops from the lids.
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Transfer each lysate with ethanol (~675 µl) into a new Viral Spin Column.
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Centrifuge the columns at ~6,800 x g for 1 minute. Discard the collection tubes with the flow-through.
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Place the Viral Spin Columns in clean Wash Tubes and add 500 µl Wash Buffer (WII) with ethanol to the Viral Spin Columns.
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Centrifuge the columns at ~6,800 x g for 1 minute. Discard the flow-through and place the spin columns back into the Wash Tubes.
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Add 500 µl Wash Buffer (WII) with ethanol into the spin columns.
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Centrifuge the columns at ~6,800 x g for 1 minute. Discard the Wash Tubes containing the flow-through.
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Place the spin columns into clean Wash Tubes.
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Centrifuge the columns at maximum speed in the microcentrifuge for 1 minute to dry the membranes completely. Discard the Wash Tubes with the flow-through.
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Place the Viral Spin Columns in clean 1.5 ml Eppendorf DNA LoBind tubes.
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Add 50 µl of nuclease-free water into the centre of each of the spin columns and close the lids.
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Incubate at room temperature for 1 minute.
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Centrifuge the spin columns at maximum speed for 1 minute. The Eppendorf DNA LoBind tubes will contain the extracted rAAV DNA for each sample. Remove and discard the spin columns.
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Quantify 1 µl of eluted sample using the ssDNA and dsDNA HS Qubit Assay kit and Qubit fluorometer.
Note: The carrier RNA in PureLink™ kit may affect the ssDNA Qubit measurements.