Sep 11, 2025
2nd generation Lentiviral production
- Hannah Lucas-Clarke1
- 1UCL/The Francis Crick Institute
Protocol Citation: Hannah Lucas-Clarke 2025. 2nd generation Lentiviral production. protocols.io https://dx.doi.org/10.17504/protocols.io.ewov11z4ovr2/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: September 11, 2025
Last Modified: September 11, 2025
Protocol Integer ID: 227021
Keywords: lentivirus for future transduction, 2nd generation lentiviral production, lentiviral production, lentivirus, plasmid, grna, future transduction, grna of interest
Abstract
This is a protocol to amplify and purify plasmids (with gRNA of interest) and package into lentivirus for future transduction.
Materials
Equipment
Centrifuges/Ultracentrifuge (RT and 4 degree)
Tissue Culture Hood appropriate for viral work
Bunsen burner
37 degree incubators one static and one shaking
Materials
Single-gRNA Expression Lentiviral Vector
pxPAX2 (Addgene, #12260)
pMD2.G (Addgene, #12259)
Plastic loops
Agar plates with the appropriate antibiotic
Ampicillin/Or other appropriate antibiotic
Liquid Broth
Large plastic centrifuge tubes
PureLink Maxi-Prep kits
Isopropanol
Ethanol
TE bufffer
HEK 293T cells
FuGene HD (Promega, cat. no. E2311)
DMEM (Gibco, cat. no. 31966021)
FBS
Pen-Step (cat. no. 15070063)
Glutamax (cat. no. 35050038)
10cm cell culture dishes
Falcon and Eppendorf tubes
0.45um filters
PBS
Troubleshooting
Vector Design
Design Mammalian Single-gRNA Expression Lentiviral Vector. Typically we use Vector Builder which deliver E.Coli stocks.
Bacteria growth on agar plates
1d
For this you need plastic loops, bunsen burner and agar plates with the appropriate antibiotic based on vector design.
Sterilise plastic loop over bunsen flame right before dipping into bacteria stock
Dip into stock and streak onto agar plate. Make sure the agar plate is labelled!
Incubate agar plate overnight at 37 degrees for bacteria to grow in colonies.
Liquid Broth
1d
Add 500uL of ampicillin (or appropriate antibiotic) to bottle of LB broth, place LB broth into large glass flask (+1 extra for control)
Remove agar plates from 37 degrees and check for successful growth of colonies
Take a pipette tip and sterilise over bunsen burner flame, pick colony and place into flask - cover flask with foil (loosely)
As a control take a tip, sterilise and place into LB flask without picking a colony
Incubate flasks shaking at 37 degrees overnight
Maxi-Prep
2h
Large plastic tubes that fit into the centrifuge are needed for this step. Remove the flasks from the incubator - there should be visible growth of bacteria in all but control flask. Transfer contents of flask into plastic centrifuge tube (without the tip)
Weigh each tube - add PBS to make up difference (this is to ensure the centriguge is balanced. Centrifuge at 4000g 4 degrees for 10 minutes.
We use the PureLink Maxi-Prep kits from Thermo Fisher Scientific. Place the red plastic top onto a glass beaker and then the column on top. Add 30mL of EQ1 buffer to each column.
Remove platic tubbes from centrifuge - discard supernatant. Resuspend sediment in 10mL of R3 buffer and pipette up and down until cloudy.
Add 10mL of L7 lysis buffer to the tube - invert to mix 4-5 times. Incubate for 5 minutes
Add 10mL of N3 buffer and shake. Transfer contents to a labelled falcon tube and centrifuge 12000g for 10 minutes (or 4000g for 30 minutes)
Remove falcons from centrifuge - there should be clear layers. Pipette liquid into column, avoid top layer and bottom layer by tilting tube. Allow to filter through column
Add 60mL wash buffer to each column. Flow through can be discarded.
Take washed column and place on top of a labelled falcon tube. Add 15mL elution buffer to each column. Once eluted - throw column.
Add 10.5mL isopropanol to each falcon - shake and centrifuge for 30 minutes at 12000g at 4 degrees.
DNA should have pelleted at base of falcon. Discard supernatant. Add 5mL 70% ethanol to each DNA pellet. Further centrifuge for 10 minutes to remove any remaining bacteria.
Remove all liquid and allow pellet to air dry for 10 minutes.
Add 200-500uL TE buffer to resuspend DNA pellet, transfer to eppendorf. Nanodrop DNA to get the concentration. Store in freezer.
Lentiviral Production
1w
Prior to this make sure you have HEK 293T cells in culture. Media: 500mL DMEM, 10% FBS, 1% Pen-Step, 1% Glutamax. When confluent, plate into 10cm cell culture dish. Good to do this on a Friday so they are 80% confluent on a Monday ready for transfection. Do multiple dishes per plasmid - i.e. 3 dishes of HEK. Remember to scale the rest of the protocol depending o the number of dishes you are doing.
3-4 hours before transfection change media on HEKs (10mL) - aim for transfection to be in the afternoon (4pm)
Add 18uL FuGene HD to 300uL DMEM and incubate for 5 minutes.
Add 3ug transfer plasmid with 2ug psPAX2 and 1ug pMD2.G in 300uL of DMEM
Mix both reactions gently and incubate for 30 minutes at room temp
Add mixture (600uL) to HEK cells
18 hours after the transfection replace the media
Turn on Ultracentrifuge - set to 4 degrees.
Harvest the media after 48 hours into 50mL falcons (curve bottom) and centrifuge 1500rpm for 3 minutes at room temperature
Filter supernatant through 0.45um filter
Transfer to ultracentrifuge tubes. Weigh to make sure this is balanced.
Centrifuge for 3 hours at 48000g
Pellet will be hard to see - circle when you remove from centrifuge. Remove supernatant in the hood and resuspened pellet in ice cold sterile PBS. 3 dishes - 850uL of PBS. Leave these on ice in fridge/cold room overnight.
Next day aliquot virus - snap freeze and transfer to -80.