PEI Transfection

PEI Transfection

Transfection:

1. Split 293T cells one day before transfection in DMEM/10% FBS medium:
   1. 6 well dish: 0.5x10⁶ cells
   2. 10cm dish: 4.0x10⁶ cells
   3. 15cm dish: 9.0x10⁶ cells

1. Prior to transfection bring all reagents to room temperature.

1. 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

1. 6 well dish: 200ul + 3 ug of total DNA
2. 10cm dish: 1mL + 7-8 ug of total DNA
3. 15cm dish: 2mL +  11-12 ug of total DNA

1. 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).

1. 6 well dish: 9ul of PEI(1ug/ul) = 9ug
2. 10cm dish: 21ul of PEI (1ug/ul) = 21ug
3. 15cm dish: 33ul of PEI(1ug/ul) = 33ug

1. Incubate 15 minutes at RT
2. Add DNA/PEI mixture to cells

1. 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(guī)格	品牌	備注
  23966-2	POLYETHYLENIMINE LINEAR	2864	2 g	polysciences	大量現(xiàn)貨
  24765-2	POLYETHYLENEIMINE 'MAX'	3184	2 g	polysciences	轉(zhuǎn)染效率高30% 更好溶解
  78002qf01	線性PEI轉(zhuǎn)染液	200	1ml	78bio	23966配制
  78002qf02	線性PEI轉(zhuǎn)染液	600	50ml	78bio	23966配制
  78002qf03	線性PEI轉(zhuǎn)染液	1800	500ml	78bio	23966配制
  78003qf01	線性PEI轉(zhuǎn)染液	200	1ml	78bio	24765配制
  78003qf02	線性PEI轉(zhuǎn)染液	700	50ml	78bio	24765配制
  78003qf03	線性PEI轉(zhuǎn)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