https://eln.excellgen.com/index.php?action=history&feed=atom&title=Lentivirus-Production-293T 2026-10-18T05:43:15Z Revision history for this page on the wiki MediaWiki 1.44.0 https://eln.excellgen.com/index.php?title=Lentivirus-Production-293T&diff=6015&oldid=prev 2012-09-12T13:10:42Z

<p></p> <p><b>New page</b></p><div>1. 24 hours before transfection, split cells into&amp;nbsp; four 15cm plates/dishes for a confluency of ~80% the next day, add 25 ml of complete DMEM medium (10% FBS, with P/S). <br /> <br /> &lt;br&gt; <br /> <br /> 2. Transfection <br /> <br /> &lt;br&gt; &#039;&#039;&#039;For each plate:&#039;&#039;&#039; <br /> <br /> In a 50 ml sterile tube, add &#039;&#039;&#039;180 ul&#039;&#039;&#039; GeneExpresso Max into &#039;&#039;&#039;600 ul&#039;&#039;&#039; serum-free&amp;nbsp; DMEM medium---&amp;gt;&#039;&#039;&#039;Mix A&#039;&#039;&#039; <br /> <br /> &lt;br&gt; <br /> <br /> In a 50 ml of sterile tube, add &#039;&#039;&#039;30 ug &#039;&#039;&#039;of lentivirus vector with GOI, then &#039;&#039;&#039;30 ug V+G&#039;&#039;&#039; packaging vector mix (V:G, &lt;strike&gt;1:2,&lt;/strike&gt; or 1:1) to &#039;&#039;&#039;600 ul&#039;&#039;&#039; DMEM medium---&amp;gt;&#039;&#039;&#039;Mix B&#039;&#039;&#039;. <br /> <br /> &lt;br&gt; <br /> <br /> Mix by pipetting using a filtered tip.&#039;&#039;&#039;Transfer Mix A to Mix B,&#039;&#039;&#039; mix by pipetting. Incubate &#039;&#039;&#039;15-20 minutes&#039;&#039;&#039; at room temperature. Pipette transfer ~&#039;&#039;&#039;1.2 ml &#039;&#039;&#039;mix onto one plate. <br /> <br /> &#039;&#039;&#039;4 plates:&#039;&#039;&#039; <br /> <br /> 720 ul GeneExpresso Max in 2.4 ml serum free DMEM <br /> <br /> 120 ug of lentivirus vector + 120 ug V+G packaging mix in 2.4 ml serum free DMEM. <br /> <br /> &lt;br&gt; <br /> <br /> 3. &lt;u&gt;&#039;&#039;&#039;Optional&#039;&#039;&#039;&lt;/u&gt; Replace medium &#039;&#039;&#039;48 hours&#039;&#039;&#039; after transfection (do not discard the medium, put the conditioned medium into clean 50 ml polypropylene tubes). Store conditioned medium in cold room. Use&amp;nbsp; medium containing &#039;&#039;&#039;2% serum &#039;&#039;&#039;and 6 μM sodium butyrate&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> 4. Collect supernatant&#039;&#039;&#039;2 and&amp;nbsp; 4 days&#039;&#039;&#039; after transfection, store virus containing-medium in cold room. Add &#039;&#039;&#039;25 ml&#039;&#039;&#039; fresh complete medium. <br /> <br /> or collect supernatant on &#039;&#039;&#039;day 3&#039;&#039;&#039;&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> &lt;strike&gt;5. Collect supernatant &#039;&#039;&#039;6 days &#039;&#039;&#039;after transfection. Discard plates.&lt;/strike&gt; <br /> <br /> &lt;br&gt; <br /> <br /> 6. [[Lentivirus Concentration PEG|Combine supernatant and precipitate the virus using PEG]]. <br /> <br /> &lt;br&gt; <br /> <br /> == Use psPAX2 (503)+ VSVG (505)&lt;br&gt; ==<br /> <br /> &lt;br&gt; <br /> <br /> {| cellspacing=&quot;1&quot; cellpadding=&quot;1&quot; border=&quot;1&quot; style=&quot;width: 436px; height: 155px;&quot;<br /> |-<br /> | &lt;br&gt; <br /> | 15cm dish <br /> | 10cm dish <br /> | 6 well<br /> |-<br /> | psPAX2 (packaging plasmid)&lt;br&gt; <br /> | 15 ug <br /> | 6 ug&lt;br&gt; <br /> | 1 ug&lt;br&gt;<br /> |-<br /> | pVSVG (envelope plasmid) <br /> | 7.5 ug&lt;br&gt; <br /> | 3 ug&lt;br&gt; <br /> | 0.5 ug&lt;br&gt;<br /> |-<br /> | pLVX or pLenti&lt;br&gt; <br /> | 22.5 ug&lt;br&gt; <br /> | 10 ug&lt;br&gt; <br /> | 1.7 ug&lt;br&gt;<br /> |-<br /> | Total DNA&lt;br&gt; <br /> | 45&lt;br&gt; <br /> | 19&lt;br&gt; <br /> | 3.2&lt;br&gt;<br /> |}<br /> <br /> &amp;nbsp;&lt;br&gt; <br /> <br /> &lt;br&gt; <br /> <br /> [[Category:Protocols]]</div>

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