Plasmids preparation and lentiviral production

We used QIAGEN’s HiSpeed Plasmid Kit (Cat# 12643) and QIAprep Spin Miniprep kit (Cat# 27104) for plasmid extraction. 

Day 0 Transfection (6-well format)

• Split cells the day before for a goal density of 70-80%.  1:3 to 1:2 for a confluent dish

• Warm OPTIMEM to RT

• Aliquot 100 microliter OPTIMEM into Eppendorf tube

• Add 2 microgram DNA to OPTIMEM.  Invert to mix and spin down. 

• Add 8 microliter PEI to the middle of the DNA/OPTIMEM solution.  Do not touch side of tube. 

• Invert several times and shake down or VERY BRIEF quick spin.  (I shake down)

• Allow complexes to form at RT for 15 min.  (I turn off lights in hood)

• For each transfection, pipet DNA/PEI solution up and down twice and add dropwise to cells. Distribute evenly.  Swirl plate and shake up/down/left/right.  Move to next transfection.

• Incubate in TC incubator for 16 hr and change media.

• For 6cm dishes everything is 2.5 times of 6 well plates, and for 10cm dishes everything is 7 times of 6 well plates.

Day1 (16 hours after transfection)

• Aspirate medium from transfected 293le dishes & feed with fresh medium

Day4

• Collect lentivirus medium from 293le dishes into conical tubes & spin down at 1200G for 5 minutes at 4°C
• Pour supernatant into syringe (prerinse the filter use 2ml Media) and push through a Millex 0.45 um PES filter disc into a new conical tube
• Add 4X LENTI-X solution (e.g. add 600uL of Lenti-X soln to 1.8mL of viral supernatant)
• Store lentivirus+LENTI-X tubes in 4°C refrigerator several hours
• Spin down lentivirus+LENTI-X tubes at 1500G for 45 minutes at 4°C
• Aspirate supernatant & resuspend pellet with a tenth of the original volume of cold PBS and store in -80°C freezers