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您的位置: 首页 >技术文献 >分析方法 >线性PEI一种高效的阳离子聚合物转染试剂

线性PEI一种高效的阳离子聚合物转染试剂

提供者:上海徕创生物科技有限公司   发布时间:2019/7/12  阅读次数:371次   进入该公司展台

基因转染实验相信实验室的大多数小伙伴都有做过,目前, 将外源DNA导入哺乳动物细胞的方法大致可分为两大类,即生物方法和物理化学方法。生物方法主要是以病毒作为载体,通过病毒感染的方式将外源DNA 导入到细胞中,物理化学方法常用的有磷酸钙法、显微注射、电穿孔、脂质体,以及高分子聚合物为载体的基因转染技术。

本文介绍的是一种阳离子高分子聚合物-------聚乙烯亚胺(Polyethyleneimine,PEI),其是一种具有较高阳离子电荷密度的有机大分子,它每第三个原子都是质子化的氨基氮原子,使得该聚合物在任何pH下都能充当有效的质子海绵体。它能将DNA缩合成带正电荷的微粒,这些微粒可以黏合到带有负电荷的细胞表面残基,并通过胞吞作用进入细胞。聚合物进入细胞后,胺的质子化导致反离子大量涌入以及渗透势降低,上述变化导致的渗透膨胀使囊泡释放聚合物与DNA形成的复合物进入细胞质,复合物拆解后,DNA就能自由的融合到细胞核中。PEI是历史上继多聚赖氨酸之后发现的第二种聚合物转染试剂。聚乙烯亚胺有线性和支链两种,有多种分子量可选,实验证明线性和支链都具有转染效果,而分MW为25000的线性PEI具有较高的转染效率。后期科研人员又对该分子进行了优化,对其分子进行了修饰,制出了MW为40000的线性PEI,使其溶解性显著提高,转染效率更高,细胞毒性更低。线性PEI广泛适用于常见细胞系,如:HEK-293、HEK293T、Hep G2、Hela、CHO-K1、COS-1、COS-7、HIH/3T3和sf9等,PEI即使在有血清存在的情况下仍然能够高效的将核酸导入细胞,其具有优越的转染效率,重组蛋白的高表达水平,且与含血清的培养基相兼容,低细胞毒性,PEI的另外一个显著优势是较其他转染试剂使用成本极低。

产品特性

CAS号:9002-98-6

分子式:(C2H5N)x

熔点:73-75℃ 

溶解性:溶于热水、低pH冷水, 甲醇和乙醇。不溶于乙醚、丙酮和冷水。

PEI MW25000操作说明:

Transfection Reagent Preparation and Storage Recommendations,These guidelines are our recommendations based off research, testing, and customer feedback. There are alternative methods available, but we suggest following these recommendations for easy adoption and consistently high performance. Thesemethods are adapted from Cold Spring Harbor Protocols, an excellent secondary resource for transient transfection literature.

Reagent Preparation (1 mg/mL)

Materials:

1.1g PEI 25K (kingmorn Catalog# KE1075).
2.1L Milli-Q® water, water for injection (WFI), or comparable biological-grade water
3.12 M Hydrochloric Acid (HCl)
4.10 M Sodium Hydroxide (NaOH)
5.Disposable 0.1-0.2um PES vacuum sterile-filter.

6.Sterile HDPE or polypropylene storage vials for reagent aliquots. 

Equipment:

1.1L glass beaker
2.1L glass graduated cylinder
3.Calibrated pH meter
4.2 x disposable 1mL plastic pipettes
5.Stir plate
6.PTFE coated stir bar
7.Vacuum pump
Method:
1.Dispense 1g of PEI 25K into beaker and suspend in 900 mL of water.
2.Add stir bar and set stirring to produce a small vortex.
3.Add hydrochloric acid dropwise until pH is< 2.0.
4.Cover top of beaker and stir for up to 3 hours until powder fully dissolves.
5.Add sodium hydroxide dropwise until pH is 6.9-7.1.
6.Transfer solution to graduated cylinder and add water until the total volume is 1L.
7.Sterile-filter through vacuum membrane.

8.Aliquot as desired and store at -20℃.

Reagent Storage:
Frozen aliquots can be stored at -20°C for up to one year. Aliquots can be thawed and kept at 4°C for up to two weeks, but should never be re-frozen.
References
1. Polyethylenimine (PEI), linear (1 mg/mL). Cold Spring Harbor Protocols 2008, pdb.rec11323– pdb.rec11323 (2008). doi:10.1101/pdb.rec11323

PEI MW4000操作说明:

Transfection Reagent Preparation (1 mg/mL)

Materials:

1.1g PEI MAX 40K (kingmorn Catalog#KE1098)

2.1L Milli-Q® water, water for injection (WFI), or comparable biological-grade water

3.1N Sodium Hydroxide, USP

4.Sterile 25 mL plastic pipette

5.0.1µm, 0.2µm, or 0.22µm PES vacuum sterile-filter unit

6.Sterile HDPE or polypropylene storage vials

Equipment:

1.1L glass beaker

2.PTFE-coated stir bar

3.Magnetic stir plate

4.1L glass graduated cylinder

5.pH meter

6.Pipette controller

7.Vacuum pump with tubing

Method:

1.In 1L glass beaker, suspend 1g of PEI MAX 40K in 900 mL water.

2.Add PTFE-coated stir bar to 1L glass beaker and set stirring to produce small vortex.

3.Wait for PEI MAX 40K to completely dissolve. This typically takes less than 5 minutes.

4.Use 25 mL plastic pipette to add 1N sodium hydroxide dropwise to 1L glass beaker until pH is 6.90 to 7.10.

5.If pH accidently exceeds 7.10, use 1N hydrochloric acid and a new 25 mL plastic pipette to lower pH back to 6.90 to 7.10.

6.Decant solution from 1L glass beaker to 1L graduated cylinder.

7.Add water to 1L graduated cylinder to adjust final volume to 1L.

8.Sterile-filter solution through vacuum membrane.

9.Aliquot solution as desired.

10.Store aliquots at 4°C

Transfection Solution Storage and Shelf Life

At 4°C in a suitable container, the transfection solution will retain its maximum performance for at least six months. For qualitative work, the solution may be suitable for use for up to a year.

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