https://eln.excellgen.com/index.php?action=history&feed=atom&title=Adenovirus_Purification_by_Ultracentrifugation 2026-10-18T02:19:36Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=Adenovirus_Purification_by_Ultracentrifugation&diff=1953&oldid=prev 2011-10-05T22:38:54Z

<p></p> <p><b>New page</b></p><div>== Initial Step Gradient ==<br /> <br /> 1. In an SW40 polyallomer centrifuge tube, add 2.5 mL of 1.4 g/mL of CsCl. <br /> <br /> 2. Add 2.5 mL of 1.25 g/mL of CsCl by placing tip of a 5-mL pipette slowly dispensing solution to make two phases. <br /> <br /> 3. Gently add ~7 mL of cleared vector supernatant on top of 1.25 g/mL CsCl. Leave about 0.5 cm at the top of the tube. <br /> <br /> 4. Add 0.5 mL of mineral oil on top of cleared vector supernatant. <br /> <br /> 5. Balance tubes against closest sample and load in rotor. Centrifuge for 90 min at 35,000 rpm, 18°C. <br /> <br /> 7. Remove tubes from the rotor with forceps. <br /> <br /> 8. Adenovirus appears as an opaque band at interface of 1.25 g/mL and 1.4 g/mL CsCl. Remove band by piercing the tube about 1 cm below vector with 2-mL syringe loaded with 18-G needle <br /> <br /> == Second Isopycnic Gradient ==<br /> <br /> 1. Add 5–6 mL of 1.34 g/mL of CsCl to the recovered vector band from previous step into a new polyallomer centrifuge tube. Cover with ~0.5 mL of mineral oil. <br /> <br /> 2. In a second tube add CsCl (1.34 g/mL) to equilibrate tubes. Centrifuge for 18 h at 35,000 rpm, 18°C. <br /> <br /> 3. Remove tube from rotor and place in black safety tube holder.&lt;br&gt;Adenovirus r appears as opaque band near the center of the tube. Remove band as above in less than 2 mL. Collect vector by keeping the volume to a minimum. <br /> <br /> == Desalting Column and Storage ==<br /> <br /> 1. Prepare PD-10 column following manufacturer’s instructions. Load up to 2 mL of vector on column.&lt;br&gt;<br /> <br /> 2. Collect by adding 0.5 mL of PBS 1× Ca++/Mg++. Repeat the step 9–10 times. Label 0.5-mL tubes.&lt;br&gt;<br /> <br /> 3. The vector is clearly visible as an opaque elute in a final volume of ~2.0–3.5 mL (from aliquots 4 to 7), depending on the initial volume size.&lt;br&gt;<br /> <br /> 4. Combine the most opaque tubes (excluding the extremities which can be used to isolate DNA for further analysis) and add glycerol to a final concentration of 10%. Another option is to dialyze the vector in several changes of PBS at 4°C.&lt;br&gt;<br /> <br /> 5. Aliquot (10, 50, and 100 uL) in 0.5-mL tubes and store at −80°C as quickly as possible.&lt;br&gt; <br /> <br /> [[Category:Protocols]]</div>

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