[實驗技術 ] PEG濃縮法 （Lentivirus 非超高速離心法）

PEG 濃縮法 (up t0 10 ~100 folds)

5x PEG8000 solutions (50 g PEG 8000/ 8.733 g NaCl in 200 mL MQ)

1. Transfer supernatant to a sterile vessel and add 1 volume of cold PEG-it Virus Precipitation Solution (4ºC) to every 4 volumes of Lentivector-containing supernatant. (Example: 5ml PEG-it with 20ml viral supernatant).

*100 mm dish= 10 mL

2. Refrigerate overnight (4ºC, at least 12 hours). Lentivectorcontaining supernatants mixed with PEG-it Virus Precipitation Solution are stable for up to 4-5 days at 4°C.

3. Centrifuge supernatant/PEG-it mixture at 1500 × g for 30 minutes at 4ºC. After centrifugation, the Lentivector particles may appear as a beige or white pellet at the bottom of the vessel.
4. Transfer supernatant to a fresh tube. Spin down residual PEGit solution by centrifugation at 1500 × g for 5 minutes. Remove all traces of fluid by aspiration, taking great care not to disturb the precipitated Lentiviral particles in pellet.

5. Resuspend/ combine lentiviral pellets in 1/10 to 1/100 of original volume using cold, sterile Phosphate Buffered Saline (PBS) or DMEM containing 25mM HEPES buffer at 4ºC.
(Example: 20ml viral supernatant-->2 mL or 200 uL).

6. Aliquot into cryogenic vials and store at -70°C until ready for use.