PEI Transfection
更新時間:2015-12-25 點擊次數:4369
PEI Transfection
Transfection:
- Split 293T cells one day before transfection in DMEM/10% FBS medium:
- 6 well dish: 0.5x106 cells
- 10cm dish: 4.0x106 cells
- 15cm dish: 9.0x106 cells
- Prior to transfection bring all reagents to room temperature.
- In a sterile tube dilute total plasmid DNA (ug) in serum-free DMEM w/o phenol red (volume of media is 10% of final volume in culture vessel). Use transgene: viral packaging (psPAX2):viral envelope (pMD2G) constructs at 4:2:1 DNA ratio
- 6 well dish: 200ul + 3 ug of total DNA
- 10cm dish: 1mL + 7-8 ug of total DNA
- 15cm dish: 2mL + 11-12 ug of total DNA
- Add PEI (1ug/uL) to the diluted DNA. Mix immediay by vortexing or pipeting. The volume of PEI used is based on a 3:1 ratio of PEI (ug):total DNA (ug).
- 6 well dish: 9ul of PEI(1ug/ul) = 9ug
- 10cm dish: 21ul of PEI (1ug/ul) = 21ug
- 15cm dish: 33ul of PEI(1ug/ul) = 33ug
- Incubate 15 minutes at RT
- Add DNA/PEI mixture to cells
- Harvest transfected cells and/or viral supernatant at 48 hours post-transfection
Reagents:
PEI (1ug/ul) – PEI is Polyethylenimine 25kD linear from Polysciences (cat# 23966-2). To make a stock solution:
- Dissolve PEI in endotoxin-free dH2O that has been heated to ~80°C.
- Let cool to room temperature.
- Neutralize to pH 7.0, filter sterilize (0.22um), aliquot and store at -20°C; a working stock can be kept at 4°C.
貨號 | 品名 | 價格¥ | 規格 | 品牌 | 備注 |
23966-2 | POLYETHYLENIMINE LINEAR | 2864 | 2 g | polysciences | 大量現貨 |
24765-2 | POLYETHYLENEIMINE 'MAX' | 3184 | 2 g | polysciences | 轉染效率高30% 更好溶解 |
78002qf01 | 線性PEI轉染液 | 200 | 1ml | 78bio | 23966配制 |
78002qf02 | 線性PEI轉染液 | 600 | 50ml | 78bio | 23966配制 |
78002qf03 | 線性PEI轉染液 | 1800 | 500ml | 78bio | 23966配制 |
78003qf01 | 線性PEI轉染液 | 200 | 1ml | 78bio | 24765配制 |
78003qf02 | 線性PEI轉染液 | 700 | 50ml | 78bio | 24765配制 |
78003qf03 | 線性PEI轉染液 | 2200 | 500ml | 78bio | 24765配制 |
78004qf01 | POLYETHYLENIMINE LINEAR | 1432 | 1g | 78bio | 23966分裝 |
78005qf01 | POLYETHYLENEIMINE 'MAX'(40 000M.W.* | 1592 | 1g | 78bio | 24765分裝 |