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Index / SBI / Lentiviral Technology pPACKH1 HIV Lentivector Packaging Kit /Product Detail : LV500A-1 Lentiviral Technology pPACKH1 HIV Lentivector Packaging Kit
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Lentiviral Technology pPACKH1 HIV Lentivector Packaging Kit

 Price: 613   EUR
695   USD
475   GBP

Product name : Lentiviral Technology pPACKH1 HIV Lentivector Packaging Kit

Catalog number : LV500A-1

Quantity: 10 transfections

Availability: Yes

Supplier name : SBI

Data sheet Data sheet : Ask more or other datasheet now !

More Details about

Lenti Starter kit
Components:

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)

Protocol
Pseudovirus Production

Day 1
1. Plate 3x106 293TN cells in a fresh 10-cm plate in 10 ml of antibiotic free DMEM medium
(DMEM+FBS+Glu).

Day 2
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
manufacturer’s protocol).


Day 4


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.


Day 5


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
TransDux protocol
Day 1


1. Plate 50,000 cells per well in a 24 well plate in culture medium.


Day 2


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.


Day 5


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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