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Index / SBI / LentiStarter Kit (pPACK-H1, PEG-it, TransDux) /Product Detail : LV050A-1 LentiStarter Kit (pPACK-H1, PEG-it, TransDux)
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#LV050A-1 LentiStarter Kit (pPACK-H1, PEG-it, TransDux)

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  Price : 506   EUR
574   USD
392   GBP
2125   Zloty
67705   JPY
3902   NOK
4181   SEK
572   CHF

Product name : LentiStarter Kit (pPACK-H1, PEG-it, TransDux)

Catalog number : LV050A-1

Quantity: 2 reactions

Availability: Yes

Supplier name : SBI

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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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Pathways :
WP1011: T Cell Receptor Signaling Pathway
WP1025: B Cell Receptor Signaling Pathway
WP1043: Calcium Regulation in the Cardiac Cell
WP1046: Signaling of Hepatocyte Growth Factor Receptor
WP1048: TGF Beta Signaling Pathway
WP1049: G Protein Signaling Pathways
WP1062: Regulation of Actin Cytoskeleton
WP1064: Delta-Notch Signaling Pathway
WP1069: Integrin-mediated cell adhesion
WP107: Translation Factors
WP1087: IL-5 Signaling Pathway
WP1091: IL-2 Signaling Pathway
WP1093: Myometrial Relaxation and Contraction Pathways
WP1096: EGFR1 Signaling Pathway
WP1103: Alpha6-Beta4 Integrin Signaling Pathway
WP113: TGF Beta Signaling Pathway
WP1130: T Cell Receptor Signaling Pathway
WP1144: B Cell Receptor Signaling Pathway
WP1159: Calcium Regulation in the Cardiac Cell
WP1162: Signaling of Hepatocyte Growth Factor Receptor
WP1164: TGF Beta Signaling Pathway
WP1165: G Protein Signaling Pathways
WP1178: Regulation of Actin Cytoskeleton
WP1180: Delta-Notch Signaling Pathway
WP1185: Integrin-mediated cell adhesion

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[GLU1] 4-hydroxy-7-methoxy-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl glucoside beta-D-glucosidase 1, chloroplastic (EC 3.2.1.182) (Beta-D-glucoside glucohydrolase) (Beta-glucosidase 1) (ZmGlu1) (EC 3.2.1.21)
[arfC MJ0671] 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate reductase (DAROPP reductase) (DARP reductase) (EC 1.1.1.302) (2,5-diamino-6-(5-phospho-D-ribosylamino)pyrimidin-4(3H)-one reductase) (2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate synthase) (DARIPP synthase) (MjaRED)
[GLU1B] 4-hydroxy-7-methoxy-3-oxo-3,4-dihydro-2H-1,4-benzoxazin-2-yl glucoside beta-D-glucosidase 1b, chloroplastic (EC 3.2.1.182) (Beta-glucosidase 1b) (Taglu1b) (EC 3.2.1.21)
[H1F0 H1FV] Histone H1.0 (Histone H1') (Histone H1(0)) [Cleaved into: Histone H1.0, N-terminally processed]
[H1f0 H1fv] Histone H1.0 (Histone H1') (Histone H1(0)) (MyD196) [Cleaved into: Histone H1.0, N-terminally processed]
[HIST1H1E H1F4] Histone H1.4 (Histone H1b) (Histone H1s-4)
[BX9] DIMBOA UDP-glucosyltransferase BX9 (EC 2.4.1.202) (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one 2-D-glucosyltransferase BX9) (Protein BENZOXAZINLESS 9)
[Bx8] DIMBOA UDP-glucosyltransferase BX8 (EC 2.4.1.202) (2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one 2-D-glucosyltransferase BX8) (Protein BENZOXAZINLESS 8)
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[env] Envelope glycoprotein gp160 (Env polyprotein) [Cleaved into: Surface protein gp120 (SU) (Glycoprotein 120) (gp120); Transmembrane protein gp41 (TM) (Glycoprotein 41) (gp41)]
[RNASEH1 RNH1] Ribonuclease H1 (RNase H1) (EC 3.1.26.4) (Ribonuclease H type II)
[RIB7 CAGL0F05203g] 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate reductase (DAROPP reductase) (DARP reductase) (EC 1.1.1.302) (2,5-diamino-6-(5-phospho-D-ribosylamino)pyrimidin-4(3H)-one reductase) (2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate synthase) (DARIPP synthase) (CglRED)
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[Arhgef2 Kiaa0651 Lbcl1 Lfc] Rho guanine nucleotide exchange factor 2 (Guanine nucleotide exchange factor H1) (GEF-H1) (LBC'S first cousin) (Lymphoid blast crisis-like 1) (Oncogene LFC) (Rhobin)
[env] Envelope glycoprotein gp160 (Env polyprotein) [Cleaved into: Surface protein gp120 (SU) (Glycoprotein 120) (gp120); Transmembrane protein gp41 (TM) (Glycoprotein 41) (gp41)]
[env] Envelope glycoprotein gp160 (Env polyprotein) [Cleaved into: Surface protein gp120 (SU) (Glycoprotein 120) (gp120); Transmembrane protein gp41 (TM) (Glycoprotein 41) (gp41)]
[env] Envelope glycoprotein gp160 (Env polyprotein) [Cleaved into: Surface protein gp120 (SU) (Glycoprotein 120) (gp120); Transmembrane protein gp41 (TM) (Glycoprotein 41) (gp41)]
[ribD2 aq_436] 2,5-diamino-6-ribosylamino-4(3H)-pyrimidinone 5'-phosphate reductase (DAROPP reductase) (DARP reductase) (EC 1.1.1.302) (2,5-diamino-6-(5-phospho-D-ribosylamino)pyrimidin-4(3H)-one reductase) (2,5-diamino-6-ribitylamino-4(3H)-pyrimidinone 5'-phosphate synthase) (DARIPP synthase) (AaeRED)
[ARHGEF2] Rho guanine nucleotide exchange factor 2 (Guanine nucleotide exchange factor H1) (GEF-H1) (Tight junction-associated guanine nucleotide exchange factor isoform A)
[ARHGEF2] Rho guanine nucleotide exchange factor 2 (Guanine nucleotide exchange factor H1) (GEF-H1)
[Arhgef2] Rho guanine nucleotide exchange factor 2 (Guanine nucleotide exchange factor H1) (GEF-H1)
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