- Materials
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- Extracted rAAV samples
- Consumables
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- 50X annealing buffer (2.5 M NaCl, 500 mM Tris-HCl, pH 7.5)
- Qubit™ ssDNA Assay Kit (ThermoFisher, Q10212)
- Qubit 1x dsDNA HS Assay Kit (ThermoFisher, Q33230)
- Qubit™ Assay Tubes (Invitrogen, Q32856)
- 0.2 ml thin-walled PCR tubes or 0.2 ml 96-well PCR plate
- Equipment
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- Thermal cycler
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
- Optional equipment
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- Qubit fluorometer (or equivalent for QC check)
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This optional self-hybridisation step can be used to self-anneal any remaining (+) and (-) single strands of rAAV vector together before library preparation.
This step can be skipped and the library preparation started immediately as we have found higher amounts of full-length inverted terminal repeat (ITR) sequences without the annealing step.
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Prepare the 50X annealing buffer (2.5 M NaCl, 500 mM Tris-HCl, pH 7.5).
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Add 1 µl of 50X annealing buffer (2.5 M NaCl, 500 mM Tris-HCl, pH 7.5) to each rAAV sample, to reach a total volume of 50 µl.
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Transfer each sample to a clean 0.2 ml PCR tube or a PCR plate.
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In a thermal cycler, incubate the tubes at 95°C for 5 minutes before ramping down to 25°C (1 minute per 1°C).
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Optional actionQuantify 1 µl of recovered rAAV using the dsDNA and ssDNA HS Qubit assay with a Qubit fluorometer.