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

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  Price : 556   EUR
631   USD
431   GBP
2336   Zloty
74432   JPY
4290   NOK
4596   SEK
629   CHF

Product name : pPACKH1 HIV Lentivector Packaging Kit

Catalog number : LV500A-1

Quantity: 200 ul

Availability: Yes

Supplier name : SBI

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About this Product :

pPACKH1 HIV Lentivector Packaging Kit lentivectors random inserts of lentivectors are considered mot harmfull and the easiers way to get DNA into nulear DNA. More precise piggy back or talen techniques are used if random inserts are not wished. Our pPACKH1 HIV Lentivector Packaging Kit will randomly isert the DNA and since bad inserts are mostly lethal, healthy modofied cells can be studied.
http://antibody-antibodies.com/anti-elisa/Lentiviral.html

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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