Product name : LentiStarter Kit (pPACK-H1, PEG-it, TransDux)
Catalog number : LV050A-1
Quantity: 2 reactions
Supplier name : SBI
Data sheet : Ask more or other datasheet now !
Lenti Starter kit
1. pPACKH1-Plamid Packaging Mix (40μl)
2. PEG-it (5 ml)
3. TransDux (200μl at 200x)
What you will need:
1. Your Lentivector construct (3rd generation preferable)
2. HEK 293TN or FT cells and culture media
3. Tabletop low speed centrifuge (ex. Beckman GS-6R)
1. Plate 3x106 293TN cells in a fresh 10-cm plate in 10 ml of antibiotic free DMEM medium
2. The cells should be 50 to 70% confluent.
3. Transfect cells with plasmid containing gene of interest (2μg) and pPACKH1-plasmid mix
(10μg=20μl). Use Lipofectamine+Plus transfection reagent from Invitrogen (Follow
4. Harvest viral supernate (culture media) and add PEG-it at a final volume of 1:5. Example: 2.5
ml of PEG-it should be added to 10 ml of viral supernate, invert 10 times to mix well. Keep
everything cold from this point onwards. Store viral Sup+ PEG-it at 40C overnight to 3 days.
5. Harvest PEG-it precipitated virus by centrifuging at 40C at 1500xg for 30 min. Aspirate off the
supernate and resuspend the milky-white pellet in a small volume (30 to 50μl) of cold sterile
PBS or cold DMEM.
6. Freeze virus at -800C.
Transduction of Target Cells
1. Plate 50,000 cells per well in a 24 well plate in culture medium.
2. Cells should be between 50 to 70% confluent.
3. Aspirate medium from cells.
4. Add TransDux to complete medium to a final concentration of 1X (Example; add 60μl of
200x TransDux to 12 ml of medium – transfer 0.5ml of this mixture per 24 well)
5. Add virus to each well at different MOIs or different volumes.
6. 72 hours post transduction, the viral genome will be integrated into the host cell genome.
7. Look at the cells for reporter expression if the viral construct has a reporter like GFP and/or
begin appropriate antibiotic selection to establish stable cell line.
OPTIONAL – Virus Titering
1. Aspirate off medium. Wash each well with PBS (at this point the plate can be frozen at -800C).
2. Add 100μl of Lysis Buffer (SBI’s Ultra Rapid Titer Kit) to each well.
3. Titer virus according to protocol given in the Ultra Rapid Titer Kit (SBI cat# LV960A-1).
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