PEI Transfection

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PEI Transfection

 

 

Transfection:

 

  1. Split 293T cells one day before transfection in DMEM/10% FBS medium:
    1. 6 well dish: 0.5×106 cells
    2. 10cm dish: 4.0×106 cells
    3. 15cm dish: 9.0×106 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.
  • 货号 品名 价格￥ 规格 品牌 备注
    23966-2 POLYETHYLENIMINE LINEAR 2864 2 g polysciences 大量现货
    24765-2 POLYETHYLENEIMINE 'MAX' 3184 2 g polysciences 转染效率高30% 更好溶解
    78002qf01 线性PEI转染液 200 1ml jinpanbio 23966配制
    78002qf02 线性PEI转染液 600 50ml jinpanbio 23966配制
    78002qf03 线性PEI转染液 1800 500ml jinpanbio 23966配制
    78003qf01 线性PEI转染液 200 1ml jinpanbio 24765配制
    78003qf02 线性PEI转染液 700 50ml jinpanbio 24765配制
    78003qf03 线性PEI转染液 2200 500ml jinpanbio 24765配制
    78004qf01 POLYETHYLENIMINE LINEAR 1432 1g jinpanbio 23966分装
    78005qf01 POLYETHYLENEIMINE 'MAX'(40 000M.W.* 1592 1g jinpanbio 24765分装