Third-Generation Lentivirus Packaging and Infection Concentration Kit
Concentrate 10–100-fold to easily obtain a high titer.
One-click copy of information
Instruction manualPrice:
¥ 2000
Item Number:
100-508
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Specification
- 5T
- 50T
隐藏域元素占位
- Product Description
- Composition
- Precautions
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Product Overview:
The third-generation lentiviral packaging, infection, and concentration kit comprises a biosafety‑enhanced mixture of third‑generation lentiviral packaging helper plasmids, a DNA transfection reagent, a viral infection enhancer, and a viral concentration solution. By employing third‑generation lentiviral vectors for virus packaging, expression clones carrying the gene of interest or shRNA can be packaged into transduction‑competent viral particles. The resulting viral particles efficiently mediate the expression of the target gene or shRNA in mammalian cells.
This product offers high safety, ease of use, rapid packaging, and a high viral titer, along with the advantage of providing a complete, one-stop kit of all necessary reagents. One kit is sufficient to produce virus in 5 × 10 cm cell culture dishes or 25 × 35 mm cell culture dishes using HEK293T cells. Unlike most commercially available viral infection kits, this product includes an infection enhancer, a concentrated solution, and a corresponding protocol—eliminating the need for additional purchases and delivering a comprehensive, end-to-end set of reagents for lentiviral packaging and infection in a single package.
Essential reagents and cells to be provided by the user
1. HEK293T cells and their corresponding culture medium. We recommend using DMEM supplemented with 10% serum.
2. Third-generation lentiviral shuttle plasmids carrying the gene of interest or shRNA. These plasmids must be purified using an endotoxin-free plasmid extraction kit; we recommend the Albatross Endotoxin-Free Plasmid Extraction Kit (Catalog No. 400-201).
Instructions for Use
1. Transfection of HEK293T cells
1.1. Cell seeding: The day before transfection, seed approximately 2.5 × 10^6 cells in a 10-cm cell culture dish and incubate in DMEM supplemented with 10% serum for 18–22 hours. Do not change the medium prior to transfection.
1.2. Plasmid Transfection:
a. Prepare two sterile 1.5 mL centrifuge tubes, and first add 300 μL of serum-free DMEM culture medium to each.
b. Add 5 µg of a lentiviral shuttle plasmid carrying the gene of interest or shRNA to 300 µL of culture medium in tube #1, then add 10 µL of the packaging helper plasmid mixture (Component 100-508A). Vortex for 10 seconds or pipette up and down 20 times.
c. Add 20 μL of DNA transfection reagent (Component 100-508B) to 300 μL of culture medium in centrifuge tube No. 2, vortex for 10 seconds, or pipette up and down 20 times.
d. After allowing the two tubes to stand at room temperature for 3 minutes, transfer the liquid from tube No. 2 into tube No. 1 (do not reverse the order of addition!), vortex vigorously for 30 seconds or pipette up and down 20 times, then let stand at room temperature for 15 minutes.
e. Using a pipette, dispense 600 μL of the mixture into the cell culture dish and gently shake to mix thoroughly.
1.3. Water Change:
After 10–16 hours of transfection, remove the old culture medium and replace it with 10 mL of fresh DMEM containing 10% serum. Discard the removed medium and pipette tips in a waste container filled with disinfectant.
2. Collect the virus:
After changing the culture medium, continue culturing for 48 to 72 hours. In a biosafety cabinet meeting the required standards, transfer the cell culture into a 15 mL centrifuge tube.
Centrifuge at 1000×g for 5 minutes, discard the pellet, and filter the supernatant through a 0.45 μm syringe filter to remove cells and debris. At this point, the viral particles in the supernatant can be directly used for titer determination, to infect target cells, or for concentration.
3. Viral Infection:
Add an appropriate amount of virus and fresh culture medium according to the MOI for the target cells, and incorporate the viral infection enhancer (Component 100-508C) at a ratio of 1:2000—specifically, add 5 μL of the enhancer per 10 mL of culture volume. First, aspirate the old medium, then add the virus diluted in 9 mL of culture medium. Next, dilute 5 μL of the viral infection enhancer in 1 mL of culture medium, distribute it evenly into the culture dish, and gently shake to mix thoroughly. After 24–48 hours of infection, replace the medium with fresh culture medium. If performing drug screening, continue culturing for an additional 12–24 hours to allow the cells to express the resistance gene.
4. Concentrated virus (optional):
Mix the lentiviral supernatant with the concentrated solution provided in the kit (Component 100-508D) at a ratio of 3:1 (3 parts supernatant to 1 part concentrate), and incubate at 4°C for 4 to 6 hours or overnight. During the initial phase, gently mix the suspension every 30 minutes. After incubation, centrifuge at 4,000 × g and 4°C for 30 minutes, then discard the supernatant. Resuspend the viral particles in DMEM or PBS at a volume equal to 1/10 to 1/100 of the original volume, gently pipette up and down to ensure uniformity, and store at…
-80℃.
5. Portioning and Storage:
Store the viral supernatant or concentrated virus aliquots at −80°C. Avoid repeated freeze–thaw cycles, as they can reduce the viral titer.
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Product Composition
Item number Component Storage conditions Specifications 100-508A Packaging helper plasmid mixture -20 ℃, one year 50 µL 100-508B DNA transfection reagent 4 ℃, one year 100 µL 100-508C Viral Infection Enhancer 4 ℃, one year 30 µL 100-508D Virus concentrate 4 ℃, half a year 20 mL -
Precautions
1. During experimental procedures, please wear a lab coat and gloves, and work within a standard‑compliant biosafety cabinet. Discard waste liquids and pipette tips in a waste container containing disinfectant.
2. This product is intended for research purposes only and shall not be used for diagnostic or therapeutic applications.
3. Only PVDF or polyethersulfone (PES) membranes may be used for filtering viral supernatants; nitrocellulose (NC) membranes are not permitted.
4. The ratio of plasmid to transfection reagent can be optimized according to the cell type; for cells that are difficult to transfect, the amount of transfection reagent may be increased.
5. The condition of the HEK293T cells used for packaging is critical to viral production; prioritize passages with a low number of subcultures. Handle with care to prevent contamination of the cell culture medium by bacteria, fungi, or mycoplasma.
6. When the shuttle plasmid carrying the target gene is too large, it can impair packaging efficiency. Once the insert exceeds 3 kb in length, the packaging efficiency declines, thereby reducing viral titer. If the size of the inserted fragment cannot be tightly controlled, it is advisable to select a smaller shuttle plasmid whenever possible.
7. The titer of the lentivirus will vary depending on the length of the target gene and the degree of cytotoxicity it imposes on the packaging cells.
8. Lentiviruses are cytotoxic to the target cells; if a large number of target cells die after virus addition, the multiplicity of infection (MOI) should be reduced, and the infection duration shortened.
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