广东快乐十分同位码:siRNA单基因套装合成 - 广东快乐十分直播开奖

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siRNA单基因套装合成

产品简介

针对靶基因设计的siRNA的基因沉默效果不尽相同。一般针对靶基因的不同区域设计三对siRNA，以确保实现靶基因的高效沉默。权阳生物将采用独有的siRNA design技术设计三对靶基因(仅人、小鼠、大鼠)siRNA，保证在标准使用条件下至少一对siRNA的mRNA水平沉默效率达70%以上。套餐产品还附送实验所需的阴性对照，阳性对照，及阳性对照引物(仅人、小鼠、大鼠)等。

套装类型

普通siRNA经济型套餐A 优惠价￥1450			普通siRNA实惠套餐B 优惠价￥1650
套餐内容	规格	纯化方式	套餐内容	规格	纯化方式
目的基因siRNA oligos	3对（3×2 OD）	HPLC	目的基因siRNA oligos	4对（4×4 OD）	HPLC
阴性对照siRNA oligos	1对（1×1 OD）	HPLC	阴性对照siRNA oligos	1对（1×1 OD）	HPLC
FAM标记阴性对照siRNA oligos	1对（1×1 OD）	HPLC	FAM标记阴性对照siRNA oligos	1对（1×1 OD）	HPLC
阳性对照siRNA oligos	1对（1×1 OD）	HPLC	阳性对照siRNA oligos	1对（1×1 OD）	HPLC

化学修饰siRNA经济套餐C 优惠价￥1750			化学修饰siRNA实惠套餐D 优惠价￥2150
套餐内容	规格	纯化方式	套餐内容	规格	纯化方式
目的基因siRNA oligos	3对（3×2 OD）	HPLC	目的基因siRNA oligos	4对（4×4 OD）	HPLC
阴性对照siRNA oligos	1对（1×1 OD）	HPLC	阴性对照siRNA oligos	1对（1×1 OD）	HPLC
FAM标记阴性对照siRNA oligos	1对（1×1 OD）	HPLC	FAM标记阴性对照siRNA oligos	1对（1×1 OD）	HPLC
阳性对照siRNA oligos	1对（1×1 OD）	HPLC	阳性对照siRNA oligos	1对（1×1 OD）	HPLC

荧光修饰siRNA经济套餐E优惠价￥2350			荧光修饰siRNA实惠套餐F 优惠价￥2850
套餐内容	规格	纯化方式	套餐内容	规格	纯化方式
目的基因siRNA oligos	3对（3×2 OD）	HPLC	目的基因siRNA oligos	4对（4×4 OD）	HPLC
阴性对照siRNA oligos	1对（1×1 OD）	HPLC	阴性对照siRNA oligos	1对（1×1 OD）	HPLC
FAM标记阴性对照siRNA oligos	1对（1×1 OD）	HPLC	FAM标记阴性对照siRNA oligos	1对（1×1 OD）	HPLC
阳性对照siRNA oligos	1对（1×1 OD）	HPLC	阳性对照siRNA oligos	1对（1×1 OD）	HPLC


in vivo 化学修饰siRNA经济套餐G优惠价￥2650			in vivo 化学修饰siRNA实惠套餐H 优惠价￥3150
（全链甲氧修饰，末端胆固醇修饰）			（全链甲氧修饰，末端胆固醇修饰）
套餐内容	规格	纯化方式	套餐内容	规格	纯化方式
目的基因siRNA oligos	3对（3×2 OD）	HPLC	目的基因siRNA oligos	4对（4×4 OD）	HPLC
阴性对照siRNA oligos	1对（1×1 OD）	HPLC	阴性对照siRNA oligos	1对（1×1 OD）	HPLC
FAM标记阴性对照siRNA oligos	1对（1×1 OD）	HPLC	FAM标记阴性对照siRNA oligos	1对（1×1 OD）	HPLC
阳性对照siRNA oligos	1对（1×1 OD）	HPLC	阳性对照siRNA oligos	1对（1×1 OD）	HPLC

实验方法

siRNA 转染
A．siRNA转染的方法
	脂质体型转染试剂进行转染需要注重的几点
哺乳动物转染的常见方法有：磷酸钙共沉淀、电穿孔法、DEAE-葡聚糖和polybrene、机械法（例如,显微注射和基因枪）、阳离子脂质体试剂，其中阳离子脂质体试剂转染法是目前最常用的转染方法。	转染试剂的用量
	siRNA的用量
	转染时的细胞密度
	转染时的操作顺序
	转染时的操作顺序


	细胞与转染试剂/siRNA复合物的温浴的时间
B．Lipofectamin2000 转染试剂
	Lipofectamin2000的应用领域
选择最适合的转染试剂和转染条件，往往取决于不同的哺乳动物细胞类型和不同的核酸分子。 Lipofectamin2000适用于核酸的体内和体外操作，可应用于DNA、RNA、反义寡核苷酸、siRNA的转染，也可应用于DNA/siRNA 的共转染操作；是一种新型的高效siRNA转染试剂。	原代培养细胞和转化细胞株的基因转染


	siRNA高通量转染试验


	DNA转染；DNA和siRNA的共转染


	核酸（siRNA、DNA、RNA）的体内导入试验


	贴壁细胞和悬浮细胞转染

	Lipofectamin2000 的特点：
	不必更换培养基，操作简便易行，可在半小时内完成操作


	在含血清培养基中也能表现高转染效率


	细胞毒性低；适用细胞广泛


	即用型试剂，可在含有抗生素的完全培养基中转染


	基于脂质的转染试剂，确保没有RNAse活性


	□ 可介导siRNA高转染细胞及体内siRNA的高效导入
C．Lipofectamin2000适用的细胞类型
Lipofectamin2000转染试剂可广泛应用于多种细胞系的DNA和siRNA转染如： HeLa(人颈部癌细胞)、MCF-7(人乳房癌细胞)、Hep3B(人肝细胞癌细胞)、COS-7(猴肾细胞)、Neuro-2a(鼠神经母细胞瘤细胞)、NIKS(人角质化细胞)、B16(鼠黑素瘤细胞)、DLD-1(人结肠癌细胞)、NIH/3T3(鼠胚胎成纤维细胞)、HT-29(人结肠腺癌细胞)、A549(人肺癌细胞)、CHO-k1(仓鼠卵巢细胞)和293(腺病毒5 DNA转化的人胚胎肾细胞),SVRbag4细胞等。

D．转染前细胞培养
					在细胞板上培养细胞时，应使细胞汇合在24小时内达到70-90%。
	细胞培养用品		表面积（mm2/孔）		细胞密度			培养基（μL /孔）
	96孔板		50		1.5´104-5.0´104			100μL
	48孔板		100		3.0´104-1.0´105			200μL
	24孔板		200		8.0´104-2.0´105			500μL
	12孔板		401		1.6´105-4.0´105			1.0 mL
	6孔板		962		3.0´105-8.0´105			2.0 mL
	35 mm		962		3.0´105-8.0´105			2.0 mL
	60 mm		2827		1.0´106-2.5´106			6.0 mL
E．合适的lipofectamin2000用量
					合适的siRNA(DNA)：lipofetamin2000比例对核酸的高效转染有重要影响；我们推荐的 DNA：lipofetamin2000为1：0.5—1：5（ug:ul），siRNA：lipofectamin2000为 1：0.01-1：0.1（pmol：ul）一般情况下，此范围内都可获得高的转染效率。
细胞培养用品		siRNA/DNA		培养基最终体积			Lipofectamin2000(siRNA/DNA)
96孔板		5pmol/0.2μg		100μL			0.25μl/0.5μl
24孔板		20pmol/0.8μg		500μL			1μl /2μl
12孔板		40 pmol /1.6μg		1 mL			2μl /4μl
6孔板		100 pmol /4.0μg		2 mL			5μl /10μl
35 mm		100 pmol /4.0μg		2 mL			5μl /10μl
60 mm		600 pmol /8.0μg		5 mL			10μl /20μl

F．贴壁细胞转染程序
	转染前一天，4-5´104细胞接种在24孔板上， 0.5mL含FBS和抗生素的DMEM（或Opti-MEM，其他培养基）细胞培养基。


选用生理状态良好的细胞对提高转染效率很	2．选择用于初期接种的细胞数量，应能在24小时内使细胞汇合达到70-90%。
重要。siRNA(DNA)和lipofectamin的用量和两者的比例可在推荐范围内适当调整。
	在50μl的DMEM（或Opti-MEM，或其他无血清培养基）无血清培养基加入20pmol siRNA(或0.8μg DNA)，柔和混匀；


	混匀lipofectamin试剂，用50μl无血清的DMEM或Opti-MEM，或其他无血清培养基）稀释1μl lipofectamin试剂（DNA转染时，则加入2μllipofectamin试剂），轻轻混匀，室温放置5分钟；


	将稀释好的siRNA和RNAi-Mate试剂混合；轻柔混匀，室温放置20分钟,以便形成siRNA/lipofectamin（或DNA/lipofectamin）复合物。


	将100μl siRNA/lipofectamin（或DNA/lipofectamin）复合物加到含有细胞和培养基的培养板的孔中，来回轻柔摇晃细胞培养板板。


	7．细胞在CO2培养箱中37℃温育24h-48h后，进行转染后的其它检测步骤。如果细胞株比较敏感，孵育4-6小时后，除去复合物，更换培养基。
G．悬浮细胞转染程序
	转染的当天，收集细胞离心，用含FBS的培养基重悬。


	在50μl的DMEM（或Opti-MEM，或其他无血清培养基）无血清的培养基加入20pmol siRNA(或0.8μg DNA)，柔和混匀；


	混匀lipofectamin试剂，用50μl无血清的DMEM或Opti-MEM，或其他无血清培养基）稀释1μl lipofectamin试剂（DNA转染时，则加入2μl lipofectamin试剂），轻轻混匀，室温放置5分钟；


	将稀释好的siRNA和lipofectamin试剂混合；轻柔混匀，室温放置20分钟,以便形成siRNA/lipofectamin（或DNA/lipofectamin）复合物。


	再加入400μL细胞悬浮液（细胞数量决定于细胞类型和转染后分析测试的时间）。


	6．细胞在CO2培养箱中37℃温育24h-48h后，进行转染后的其它检测步骤。如果细胞株比较敏感，孵育4-6小时后，除去复合物，更换培养基。





H．DNA和siRNA共转染细胞
	在转染的前一天，4-5´104细胞接种在24孔板上，0.5 mL含FBS和抗生素的细胞培养基。


	选择用于初期接种的细胞密度，应能在24小时内使细胞汇合达到70-90%。


	在100μL的无血清的培养基中稀释20pmol siRNA和0.2μg DNA，加入2μl lipofectamin试剂，充分混合，放置20分钟,以便形成siRNA/ DNA/lipofectamin复合物。


	将siRNA/ DNA/lipofectamin复合物加入培养基中，轻轻混匀。


	5．细胞在37℃温育24h-48h后，进行转染后的其它步骤。
I．siRNA体内导入方法
	适量的siRNA或DNA溶于不含RNA酶的无菌水中，轻轻混匀，因为注射液体积有限，建议采用高浓度的siRNA或DNA，一般DNA为2μg /μL、siRNA为10μg /μL。


	取适量的DNA、siRNA或siRNA\DNA复合物与lipofectamin混合。例如，在1#管中加入0.5μL的DNA（1μg）和 0.5μL的siRNA（5μg），在2#管中加入0.55μL的lipofectamin（24μg）和0.45μL不含RNA酶的无菌水中，将1#管中的溶液加入2#管中，在室温下温育30分钟，以形成siRNA/DNA-lipofectamin复合物。


	3．制备的siRNA/DNA-lipofectamine复合物可用于体内导入siRNA、DNA或siRNA\DNA。

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