{"id":273,"date":"2023-06-06T11:39:26","date_gmt":"2023-06-06T03:39:26","guid":{"rendered":"http:\/\/8.218.156.193\/?post_type=resource&p=273"},"modified":"2023-07-07T12:35:06","modified_gmt":"2023-07-07T04:35:06","slug":"protocol","status":"publish","type":"resource","link":"http:\/\/yuhongtaolab.com\/en\/resource\/protocol\/","title":{"rendered":"Lentivirus production protocol"},"content":{"rendered":"

A. Transfection in 10cm dish<\/strong><\/p>\n

    \n
  1. Day 1. Seed 293FT cell into 10cm dish, ~1e7\/well<\/li>\n
  2. Day 2. Cells grow to ~90% confluence, perform transfection with Lipofectamine 2000\n
      \n
    1. Mix 1. Dilute 50ul Lipofectamine 2000 in 1.5ml Opti-MEM, mix well, incubate for 5min at RT.<\/li>\n
    2. Mix 2. Dilute the following plasmids in 1.5ml Opti-MEM, mix well, and incubate for 5min at RT.\n
        \n
      1. 5.6ug pMD2.G<\/li>\n
      2. 14ug Target vector<\/li>\n
      3. 8.4ug psPax2<\/li>\n<\/ol>\n<\/li>\n
      4. Mix 1 and 2, then incubate for 10min at RT;<\/li>\n
      5. Aspirate old medium, add 8ml serum-free medium, and add transfection mix into cells;<\/li>\n
      6. After 4-5 hours, change to 8ml fresh complete DMEM medium.<\/li>\n<\/ol>\n<\/li>\n
      7. Day 4. Collect the 1st viral supernatant into 15ml tubes and concentrate with lenti-X concentrator, add 8ml fresh DMEM medium to cells, and continue culturing;<\/li>\n
      8. Day 5. Collect the 2nd viral supernatant and discard the packaging cell, and concentrate the 2nd viral supernatant with Lenti-X concentrator.<\/li>\n<\/ol>\n

         <\/p>\n

        B. Transfection in 6-well plate<\/strong><\/p>\n

          \n
        1. Day 1. Seed 293FT cell into 6-well plate, ~1e6\/well<\/li>\n
        2. Day 2. ~90% confluence, transfection with Lipofectamine 2000\n
            \n
          1. Mix 1. Dilute 10ul Lipofectamine 2000 in 125ul Opti-MEM, mix well, incubate for 5min at RT.<\/li>\n
          2. Mix 2. Dilute the following plasmids in 125ul Opti-MEM, mix well, and incubate for 5min at RT.\n
              \n
            1. 0.9ug pMD2.G<\/li>\n
            2. 2.3ug Target vector<\/li>\n
            3. 1.4ug psPax2<\/li>\n<\/ol>\n<\/li>\n
            4. Mix 1 and 2, then incubate for 10min at RT;<\/li>\n
            5. Aspirate old medium, add 1.5ml serum-free medium, and add transfection mix into cells;<\/li>\n
            6. After 4-5 hours, change to 2ml fresh complete DMEM medium.<\/li>\n<\/ol>\n<\/li>\n
            7. Day 4. Collect the 1st Viral supernatant into 15ml tubes and concentrate with Lenti-X concentrator, add 2ml fresh medium to cells and continue culturing;<\/li>\n
            8. Day 5. Collect the 2nd viral supernatant and discard the packaging cell, and concentrate the viral supernatant as previously described.<\/li>\n<\/ol>\n

               <\/p>\n

              Virus concentration protocol<\/strong><\/p>\n

                \n
              1. Centrifuge viral supernatant at 500g for 10min.<\/li>\n
              2. Transfer the supernatant into a new 15ml tube, and discard cell debris.<\/li>\n
              3. Add Lenti-X concentrator into viral supernatant at a 1:3 ratio, and mix by gently inverting.<\/li>\n
              4. Incubate at 4C for 30min to 24h (minimum 30 min, maximum 1 week).<\/li>\n
              5. Centrifuge at 1500g, 4\u2103 for 45min.<\/li>\n
              6. Discard supernatant.<\/li>\n
              7. Re-suspend viral pellet with 500ul-1ml PBS.<\/li>\n
              8. Aliquot into small volume and store at -80C until use.<\/li>\n<\/ol>\n

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