Editing AAV-Production-293T-2 (section)

== PREPARATION AND LOADING OF IODIXANOL GRADIENT<br>  ==

Preformed iodixanol step gradients are used to expedite the centrifugation step. The 40% and 25% iodixanol steps are used to remove contaminants with lower densities including empty capsids, and a 60% step serves as a cushion for genome-containing recombinant AAV virions. To clearly see the steps, phenol red is included in the upper 25% and lower 60% steps. The first step (15%) has 1 M NaCl to destabilize ionic interactions between macromolecules. The following volumes are for two gradients using OptiSeal tubes. The 1.27 mm x 89 mm spinal needle holds ~0.4 ml.<br>1. Prepare the following iodixanol solutions:<br>15% Iodixanol step: Mix 4.5 ml of 60% iodixanol and 13.5 ml of 1 M NaCl/PBS-MK buffer.<br>25% Iodixanol step: Mix 5 ml of 60% iodixanol, 7 ml of PBS-MK buffer and 30 ÎŒl of Phenol red.<br>40% Iodixanol step: Mix 6.7 ml of 60% iodixanol and 3.3 ml of PBS-MK buffer.<br>60% Iodixanol step: Mix 10 ml of 60% iodixanol and 45 ÎŒl of Phenol red.<br>2. Overlay each centrifuge tube with these solutions in the order below using a 10-ml syringe and 1.27 mm Ã 89 mm spinal needle, taking care to avoid bubbles. The same needle can be used for loading all steps.<br>5 ml of 60% iodixanol step<br>5 ml of 40% iodixanol step<br>6 ml of 25% iodixanol step<br>9 ml of 15% iodixanol step<br>3. Continue from Step 25 of PROCEDURE.<br> 

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