[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.

[實驗技術]PEI 細胞轉染

Polyethylenimine(PEI)轉染(Transfection)實驗

原理:
Polyethylenimine(PEI)為一個帶正電的分子，而他會以包覆的形式和DNA形成一個複合物，並且將欲送入的DNA送到細胞內表現。

60 mm dish scale (HEK 293T cells)

Day 0 晚上  細胞播種  3-4 x 10^5 cell/ dish （ 12 小時後 約80%滿）
Day 1 早上  準備轉染的Plasmid

材料 （per dish）：
Plasmid DNA 4 ug,
PEI 4 uL,
OPTI-MEM (or serum free medium) 400 uL
Mix well （ 10-15 sec x2 vortex)
靜置15 mins,
在靜置的時間內，更新細胞培養基（4 mL/dish）
以放射狀的方式，緩慢地將混合液加入細胞中，盡量避免傷害細胞
6小時後再次更換新的培養基 （觀察細胞狀態  若死細胞不多  可以隔天再換）

Day 2 Post-transfection for 24 hours (若轉染DNA載有GFP等螢光Taq, 應該可以看見      GFP訊號)
Day 3 Post-tranfection for 48 hours,
原則上轉染後，在30-48小時內表現量最大
基本上  在293T cells這類容易易轉染的細胞 約80%以上細胞有螢光訊號  轉染才算成功
若是FLAG, MYC 等非螢光蛋白  可進行西方點墨法 確認標的蛋白是否過度表現(overexpression)