Titration of Lentivirus

When aliqouting the virus, prepare 1 vial with 6 μl for titration. This vial must go through one cycle of freeze / thaw before titration and if you want to titer in conjunction with aliqouting you can put the vials on dry-ice for a bit.

Seed cells in 6 well plates seed 100,000 cells - in 2mL - 3mL. For each virus - 3x wells are required. Plate 6x additional wells for control and reference batch.

Dilute the virus 1:10 before adding it to the cells [54µL + 6µL].

Then add 3 μl, 10 μl and 30 μl of the diluted virus to the cells [corresponding to 0.3 μl, 1 μl and 3 μl undiluted virus].

Leave for 72h 0m 0s before DNA is isolated.

Look at the cells under the microscope and compare with control.

Remove media and wash 1x with 1mL [Use 1000 ul pipette and don´t add the PBS directly on the cells to avoid flushing them off].

Add 500µL and leave for a few minutes in the incubator.

After incubation with trypsin, add 500µL and transfer cells to 1.5 ml tubes.

Spin down at 300x g.

Remove supernatant with a pipette to not disturb the pellet.

Make a master mix of 200µL + 20µL per sample and resuspend the pellet in 220µL.

Add 200µL [w/o ethanol] and mix thoroughly by vortexing.

Bring samples to DNA isolation room and follow the instructions from the Qiagen DNeasy Protocol: Purification of Total DNA from Animal Blood or Cells [Spin-Column Protocol]. Start with incubation at 56°C for 0h 10m 0s and then move directly to step 3.

Assess titre using qPCR.