ESO wikiadmin: Created page with "{| class="wikitable" |+ !Material ! |- | |293T cells |- | |2x HBS-buffer |- | |2.5 M CaCl₂ |- | |25 mM Chloroquin |- | |Lentiviral vectors |} === Procedure === # A confluent plate of 293T cells is splitted 1:5 one day before transfection # The transfection reaction contains: 500 µl ddH₂O, 6.5 µg pLKO.1 (with gene or shRNA of interest), 3.5 µg pMD2.G (plasmid #1256) and 5 µg psPAX2 (plasmid #1257) # Add 500 µl 2x HBS-buffer # While vortexing t..."

2025-02-15T17:46:14Z

Created page with "{| class="wikitable" |+ !Material ! |- | |293T cells |- | |2x HBS-buffer |- | |2.5 M CaCl2 |- | |25 mM Chloroquin |- | |Lentiviral vectors |} === Procedure === # A confluent plate of 293T cells is splitted 1:5 one day before transfection # The transfection reaction contains: 500 µl ddH2O, 6.5 µg pLKO.1 (with gene or shRNA of interest), 3.5 µg pMD2.G (plasmid #1256) and 5 µg psPAX2 (plasmid #1257) # Add 500 µl 2x HBS-buffer # While vortexing t..."

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{| class="wikitable"
|+
!Material
!
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|293T cells
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|2x HBS-buffer
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|2.5 M CaCl2
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|25 mM Chloroquin
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|Lentiviral vectors
|}

=== Procedure ===

# A confluent plate of 293T cells is splitted 1:5 one day before transfection
# The transfection reaction contains: 500 µl ddH2O, 6.5 µg pLKO.1 (with gene or shRNA of interest), 3.5 µg pMD2.G (plasmid #1256) and 5 µg psPAX2 (plasmid #1257)
# Add 500 µl 2x HBS-buffer
# While vortexing the reaction, 50 µl of 2.5 M CaCl2 are added dropwise
# Incubate for 10 min at room temperature
# During the formation of Ca2+/DNA precipitates, add 10 µl of chloroquin (25 mM) to each 10 cm culture dish with cells growing in a volume of 10 ml medium
# Distribute the ~1ml of transfection mix dropwise onto the cell culture plate (carpet bombing)
# Aspirate chloroquin-containing medium after 6 h and replace with 6 ml fresh DMEM / 10% CS / Pen/Strep
# After 48-72 h, the virus-containing supernatant is collected and centrifuged for 7 min at 2000 rpm and 4°C
# After 48-72 h the virus-containing supernatant is collected and centrifuged for 7 min at 2000 rpm and 4°C
# Sterile filtration of supernatant (0.2 µm pore size) and directly employ in transduction of target cells or aliquote and quick-freeze the supernatant in liquid nitrogen, storage at -80° C

Production of lentiviral particles - Revision history