- Materials
-
- 2.6 x10^10 GC of rAAV per sample
- Consumables
-
- DNase I (NEB, M0303)
- 0.5 M EDTA (Fisher Scientific, 11568896)
- Nuclease-free water (e.g. ThermoFisher, AM9937)
- 1.5 ml Eppendorf DNA LoBind tubes
- Equipment
-
- Thermal cycler
- Extraction hood
- Microfuge
- Ice bucket with ice
- P1000 pipette and tips
- P200 pipette and tips
- P100 pipette and tips
- P20 pipette and tips
-
DNAseI treatment is carried out before extraction to remove any non-encapsidated DNA from the rAAV preparations.
-
Thaw the DNaseI reaction buffer and rAAV lysates (if they have been stored in the freezer) at room temperature and place on ice.
-
Prepare each rAAV sample in nuclease-free water:
- Transfer ≥2.6 x1010 GC of AAV sample into a 1.5 ml Eppendord DNA LoBind tube.
- Adjust the volume to 170 µl with nuclease-free water.
- Mix by pipetting up and down.
- Spin down briefly in a microfuge.
-
Combine the following reagents in the 1.5 ml Eppendorf DNA LoBind tube for each sample.
Reagent Volume rAAV sample (≥2.6 x1010 GC per sample) 170 µl DNAseI reaction buffer 20 µl DNAseI 10 µl Total 200 µl -
Thoroughly mix the reaction by gently pipetting and briefly spinning down.
-
Using a thermal cycler, incubate at 37°C for 10 minutes.
-
Add 2 µl of 0.5 M EDTA to each sample and mix thoroughly by pipetting and spin down briefly.
-
Using a thermal cycler, incubate at 72°C for 10 minutes.