|Lentiviral RNAi protocols
envelpe plasmid :VSV-g /Pmd2.G
Haipin pLKO.1 vector (TRC library plasmid)
Low –antibotic medium:0.1 x Pen/strep, DMEM +10% FBS
Viral havrset medium:30% FBS, DMEM, 1x pen/Strep
Day 0: Preparation of HEK293T packaging cells
18-24 hours prior to transfection, plate HEK293T cells on a 10 –cm plate
Day 1: Transfection
Replace with 4.5 ml low –antibiotic medium when the cells are
transfect with Roche FuGene HD Transfection reagent
for 100-mm plate with the following amount of plasmid DNA:
hairpin pLKO.1 5 ug
psPAX2 5 ug
VSV-G 2 ug( total DNA : 12 ug)
0.5 ml OPTI-MEM + DNA 12 ug ( 3 plasmids) + 40 ul Fugene HD
-> vortex and incubate at RT 20 min
-> Add the transfection mixture to the cells in a drop-wise manner.
-> Swirl then plate to ensure the mixture distributes over the entire plates surface.
Day 2: 18 hours post-transfection
Change media and replace with 5ml viral harvest medium.
Day 3: 48 hours post-transfection, viral collection
Incubate for 24 hours
Filtrate the supernantant through a filter with pore size 0.45 nm ( before using, moisten your filter with media).
Aliquot and store the 5 –ml virus supernatant at -80c degree ( avoid multiple freeze –thaws).
Add 5 ml fresh viral harvest medium to the cell cultures.
Day 4: same collection as the above.
Discard the HEK293T cell culture after bleach it.
Infecting the target Cells
Day 0: seed cells in 100-mm culture plate
Day 1: Infection
Add lentivirus to cells in growth medium containing polybrene
Remove growth medium when the cells are around 70% confluent
2. Add total 6 ml fresh medium which contains 8 ug/ml polybrene
Mix 4 ml complete DMEM and 2ml lentiviral supernatant in a 15 ml centrifuge tube, then add 9.6 ul of 5 mg/ml polybrene into the tube.
Polybrene (Hexadimethrine bromide) stock solution: 50 mg powder into 10 ml saline , then filter through 0.2 um
Day 2 Select with Puromycin
Remove growth medium
Add total 6 ml fresh medium which contains 2 ug/ml puromycin
Puromycin stock solution: Dissolve 100 mg powder in 10 ml distilled water then filter through 0.2 um . Aliqoutify and freeze at -20 C .
Day 4 Select with puromycin
Add total 6 ml fresh medium which contain 2 ug/ml puromycin *.ug: microgram
Day 6 Harvest cells for protein assay
Yue Zhang, PhD in Dr. Stuart K Calderwood lab email@example.com