线性PEI一种高效的阳离子聚合物转染试剂_分析方法_技术文章_上海...

来自 : 发布时间：2024-04-29

主营产品：厌氧产气袋,厌氧培养罐,厌氧产气包,日本三菱厌氧袋,厌氧培养盒,ZETA LIFE 胎牛血清,转染试剂,无血清培养基,生物技术服务代做阿仪网认证请放心拨打 15021202164已获得阿仪网信誉等级评定76成长值(0-40)基础信誉积累，可浏览访问(41-90)良好信誉积累，可接洽商谈(91+)优质信誉积累，可持续信赖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, andcustomer 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 water3.12 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 beaker2.1L glass graduated cylinder3.Calibrated pH meter4.2 x disposable 1mL plastic pipettes5.Stir plate6.PTFE coated stir bar7.Vacuum pumpMethod: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.References1. Polyethylenimine (PEI), linear (1 mg/mL). Cold Spring Harbor Protocols 2008, pdb.rec11323 pdb.rec11323 (2008). doi:10.1101/pdb.rec11323PEI 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 water3.1N Sodium Hydroxide, USP4.Sterile 25 mL plastic pipette5.0.1 m, 0.2 m, or 0.22 m PES vacuum sterile-filter unit6.Sterile HDPE or polypropylene storage vialsEquipment：1.1L glass beaker2.PTFE-coated stir bar3.Magnetic stir plate4.1L glass graduated cylinder5.pH meter6.Pipette controller7.Vacuum pump with tubingMethod：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 CTransfection Solution Storage ａnd Shelf LifeAt 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.原创作者：上海创凌生物科技有限公司