Editing Lentivirus-Production-293T

1. 24 hours before transfection, split cells into&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). 

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2. Transfection 

<br> '''For each plate:''' 

In a 50 ml sterile tube, add '''180 ul''' GeneExpresso Max into '''600 ul''' serum-free&nbsp; DMEM medium---&gt;'''Mix A''' 

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In a 50 ml of sterile tube, add '''30 ug '''of lentivirus vector with GOI, then '''30 ug V+G''' packaging vector mix (V:G, <strike>1:2,</strike> or 1:1) to '''600 ul''' DMEM medium---&gt;'''Mix B'''. 

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Mix by pipetting using a filtered tip.'''Transfer Mix A to Mix B,''' mix by pipetting. Incubate '''15-20 minutes''' at room temperature. Pipette transfer ~'''1.2 ml '''mix onto one plate. 

'''4 plates:''' 

720 ul GeneExpresso Max in 2.4 ml serum free DMEM 

120 ug of lentivirus vector + 120 ug V+G packaging mix in 2.4 ml serum free DMEM. 

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3. <u>'''Optional'''</u> Replace medium '''48 hours''' after transfection (do not discard the medium, put the conditioned medium into clean 50 ml polypropylene tubes). Store conditioned medium in cold room. Use&nbsp; medium containing '''2% serum '''and 6 μM sodium butyrate<br> 

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4. Collect supernatant'''2 and&nbsp; 4 days''' after transfection, store virus containing-medium in cold room. Add '''25 ml''' fresh complete medium. 

or collect supernatant on '''day 3'''<br> 

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<strike>5. Collect supernatant '''6 days '''after transfection. Discard plates.</strike> 

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6. [[Lentivirus Concentration PEG|Combine supernatant and precipitate the virus using PEG]]. 

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== Use psPAX2 (503)+ VSVG (505)<br>  ==

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{| cellspacing="1" cellpadding="1" border="1" style="width: 436px; height: 155px;"
|-
| <br> 
| 15cm dish 
| 10cm dish 
| 6 well
|-
| psPAX2 (packaging plasmid)<br> 
| 15 ug 
| 6 ug<br> 
| 1 ug<br>
|-
| pVSVG (envelope plasmid) 
| 7.5 ug<br> 
| 3 ug<br> 
| 0.5 ug<br>
|-
| pLVX or pLenti<br> 
| 22.5 ug<br> 
| 10 ug<br> 
| 1.7 ug<br>
|-
| Total DNA<br> 
| 45<br> 
| 19<br> 
| 3.2<br>
|}

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[[Category:Protocols]]