UCSF

Lentiviral Transfection

Lentiviral Transfection (10cm plate) and Concentration Protocol

Modified from McManus Lab

Materials for transfection

  1. 293T cells – 10cm plate-60-70% confluent

  2. 30 μL Fugene transfection reagent (Roche)

  3. OptiMEM (Gibco)

  4. 4 μg Master mix-equal volumes of pVSV-G, pMDL and pRSV

  5. 4 μg lentivirus construct (i.e. your GFP-shRNA construct)

Transfection protocol

  1. In 1.5 mL tubes, mix 600 μL OptiMEM with 30μL Fugene. (NOTE: Do not allow the Fugene to come in contact with the tube in its undiluted form.) Incubate for 5 mins.

  2. In the cap of the tube, mix 4μg master mix with 4μg lentivirus construct.

  3. Close lid and mix with Fugene mixture.

  4. Incubate 20 mins at R.T.

  5. Pour transfection mixture into 293T cells.

  6. Allow viral production to continue for 48-72 hrs before harvesting.

  7. Harvest and concentrate (see protocol below).

Note: Usually transfect on Friday and concentrate virus, Monday morning.

Materials for concentration

  1. Ultraclear SW41 centrifuge tubes (Beckman Cat. # 344059)

  2. 10 mL syringe

  3. 0.45 μm syringe filters (SFCA filter, Corning Cat. # 431220)

  4. 1X sterile PBS

  5. Kimwipes

  6. Ultracentrifuge with a SW41 rotor (10th floor, Diabetes Center)

Virus concentration protocol

  1. Before collecting the supernatant, turn on the vacuum of the ultracentrifuge, this helps it cool to 4°C quickly.

  2. Following transfection, check under the microscope for GFP-transfection efficiency. The transfected cells look rounder and should be GFP positive. The media will slightly change color, an indication of active cell metabolism. Using a 10ml, syringe, carefully collect media from transfected plates.

    Pay attention not to disturb the 293T monolayer of cells.

  3. Using the 0.45 μm syringe filter, filter the media into ultraclear SW41 centrifuge tubes. Pull up the piston of the syringe after filtering to avoid dripping.

    Each tube holds approximately 12ml, and it is important for the level of the liquid to be 3-5mm from the top. Bleach the syringe and filter for 45 mins. Also add 10% bleach to the petri-plates containing 293T cells.

  4. Balance the tubes and spin using a SW41 rotor (stored at 4°C, 10th floor) for 90 mins at 4°C at 25,000 rpm.

    Tubes may be balanced with serum free media. The rotor holds a maximum of 6 tubes. If you are doing 3 samples for e.g., fill a centrifuge tube with media and balance the third tube.

  5. After centrifugation, carefully decant the supernatant into 10% bleach solution. Place the tube in an inverted position with a Kimwipe blocking the mouth of the tube to collect any liquid that may drip.

  6. Resuspend the pellet with 100 μL 1X PBS and leave standing at 4°C overnight. Cover the mouth of the tube with parafilm to avoid evaporation and spilling.

    To resuspend, pipet the pellet up and down approximately 20 times, using a 200μL pipettor.

  7. Following the overnight incubation, the virus is ready to aliquote. Single use aliquots can be stored at -80°C.