Lentiviral Packaging Kit Protocol
This protocol gives a step-by-step guide on how to use the Lenti-vpack packaging kit for lentiviral particle production.
Note: Performing Lentiviral experiments may require special laboratory conditions and/or permissions (BSL2). Follow the guidelines and regulations of your institution. Perform the experiments with due caution to avoid exposure to infectious materials.
*For OriGene's Lenti shRNA application, we recommend a 6-well plate format. **The protocol below is a for a 10cm dish, see the table blow for different vessels and reagent requirements.
Vessel | Cells | Lenti Plasmids | Packaging Plasmids | Transfection Reagent | Opti-MEM | Reactionsper Kit |
---|---|---|---|---|---|---|
10-cm dish | 2.5x106 | 5 μg | 6 μg | 33 μL | 1.5 mL | 10 |
6-well plate | 5x105 | 1 μg | 1.2 μg | 6.6 μL | 250 mL | 50 |
12-well plate | 2.5x105 | 0.5 μg | 0.6 μg | 3.3 μL | 100 mL | 100 |
Day 1, Plate Cells: Plate 2.5 x 106 HEK 293T cells on a 10cm dish in 10 mL complete growth media (antibiotic-free preferred) and incubate at 37°C overnight.
Day 2, Transfection:
- In a labeled Eppendorf tube, dilute the following DNA in 1.5 mL Opti-MEM, and pipet gently to mix completely.
- 5 μg of pLenti-shRNA construct or 5 μg of pLenti-ORF expression construct
- 6 μg of packaging plasmids
- Add 33 μL of TurboFectin transfection reagent to the diluted DNA (not the reversed order), pipet gently to mix completely.
- Incubate for 15 min at room temperature.
- Add the transfection mixture prepared above, dropwise to the cells. Gently rock the plate back-and-forth and from side-to-side to distribute the complex evenly. Incubate at 37°C.
Note: With TurboFectin, no medium change is necessary, directly add the transfection mixture to cells in complete growth media.
Day 3, Change Medium: Change the culture medium after 12-18 hours of incubation.
Day 4, Harvest 1st Batch: Harvest the first batch of viral supernatant from the culture and store at 4°C. Add 10 mL fresh culture media to the cell culture.
Day 5, Harvest 2nd Batch:
- Harvest the second batch of viral supernatant and combine with the first batch.
- Filter through a 0.45μm PES filter to remove cellular debris. The viral titer at this step is usually 106 -107 TU/mL**. The viral supernatant is now ready for the majority of transduction applications. If necessary, further concentration can be applied ( lentiviral concentrator).
Note: Large ORF inserts might decrease the viral titer