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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

杂交瘤细胞筛选与克隆（DiSH™ Kit）

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杂交瘤细胞筛选与克隆（DiSH™ Kit）

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  • 相关资料
  • Q&A
  • 参考文献

杂交瘤细胞筛选与克隆（DiSH™ Kit）

杂交瘤细胞筛选与克隆（DiSH™ Kit）



　　DiSH™ Kit 是新开发的单克隆抗体筛选系统，试剂盒中含有获得专利的 Sp2ab 骨髓瘤细胞系，可以使产生的杂交瘤细胞系分泌特异性免疫球蛋白（Ig），并将抗体表达在细胞表面，表面结合抗体可以使抗原特异性杂交瘤细胞被标记。被标记的杂交瘤细胞就可通过流式细胞仪（FACS）或磁珠从成百上千的细胞中分离出来。从分选完杂交瘤细胞至获得首轮筛选结果，仅需16天，可节省75% 时间，大大提高了高度特异性单克隆抗体的检测效率。

 


◆特点

●　节约时间、减少人力成本

●　更易获得特异稳定的杂交瘤细胞

●　无需有限稀释法，降低实验工作量

术

？

什么是 DiSH™ 技术呢？

抗原特异性杂交瘤的分离包括四个步骤：

 

1. 免疫：使用靶抗原免疫小鼠

2. 融合：B细胞与多发性骨髓瘤细胞 Sp2ab 融合（Sp2ab 是 Abeome 公司获得专利的骨髓瘤细胞），形成的杂交瘤细胞可分泌并在细胞表面

2. 表达特异性免疫球蛋白。

3. 标记/富集：带有标记的抗原可捕获抗原特异性杂交瘤细胞，利用流式细胞术（FACS）或磁珠进行筛选分离。

4. 克隆：可通过以下两种方法分离和克隆抗原特异性杂交瘤细胞：

●　FACS：通过流式细胞仪筛选杂交瘤细胞

●　磁珠分离：在 AbeoClone™ 培养基（半固体琼脂）中培养细胞，筛选可见克隆

杂交瘤细胞筛选与克隆（DiSH™ Kit）

◆相关资料

Alternative Name:

Hybridoma Kit

Application Notes:

Uses:
Hybridoma generation and Cloning
Cell line development
Recommended for generation, selection and cloning of mouse monoclonal   hybridomas.

Quantity:

For fusion of 1×108 total lymphocytes

Use/Stability:

When stored properly, these reagents are stable for one   year from date received.

Handling:

Upon receipt, remove the vial of cell line from the box   and store in liquid nitrogen. Store the remaining reagents at -20°C. Avoid   repeated freezing and thawing by storing aliquots at -20°C.

Shipping:

Shipped on Dry Ice

Kit/Set Contains:

1 vial of Sp2ab myeloma partner cells
1.5mL PEG
200mL PCM (Prefusion Medium)
(2X) 200mL WCM (Wash Medium)
200mL FCM (Fusion Medium)
90mL AbeoClone™ Medium
(2X) 200mL RCM (Recovery Medium)
200mL ECM (Expansion Medium)

Technical Info/Product Notes:

Application Note:
Anti-hESC mAb Hesca-2 Binds to a   Glycan Epitope Commonly Found on Carcinomas

产品列表
产品编号 产品名称 产品规格 产品等级 备注
ENZ-71001-0001 DiSH™ kit
杂交瘤细胞筛选与克隆		 1 Kit – –