Viral prep: aliquot and titer Remove centrifuge tubes from the cold room and open parafilm. Using a P200 pipetter, pipette the PBS up and down on the bottom of the tube to resuspend the pellet. You may also gently scrape the tip on the bottom of the tube. Pipette up and down about 5-6 times, then move the 100uL to an eppendorf tube. You can combine the two pellet resuspensions for each of the three constructs. Label a bunch of eppendorf tubes with the name of each construct and the date. Pipette 20uL into each tube. Snap freeze all of the tubes on a dry ice/ethanol bath. Store all but one tube for each construct in the –80 freezer on the 4th floor autoclave room in a new box labeled lentivirus with todays date. Take the one last tube and thaw it. Make up 10mL of DMEM/10% FBS (normal 293T medium) with a 1:1000 dilution (so 10uL) of polybrene. Mix well. Pipette 50uL of this solution into each of 18 eppendorf tubes. You will use these to make 6 10-fold serial dilutions for each of the three viruses;. Add 5uL of each of the three viruses to each of three eppendorf tubes with medium. Mix thoroughly. Take 5uL from these tube with virus and add to the next three tubes in line. Mix again. Etc till all 6 tubes are done. Label the 96 well plate, with sets of 6 wells for the dilution series for each virus. Remove the medium from each well of the 96 well plate and replace with one of the viral dilution tubes. Return the plate to the incubator overnight. Next day, remove the medium from each well and replace with fresh normal 293T medium (DMEM/10% FBS). 48 hours later (60-72 hours after the initial infection), count the titer by determining which is the last well where GFP positive cells can be seen. Count the number of positive cells in that well, divide by 5 and multiply by 10 to the power of the well number (if it is the 6th well, you multiply by 10 to the 6th).