基本描述

英文名称	miRNA miniprep Kit
储存温度	室温
运输条件	常规运输
产品介绍	本试剂盒用于从各种动物组织、植物组织和细胞中提取miRNA。提取的miRNA 分子完整、纯度高，适用于Northern Blot 、Real-TimePCR、miRNA微列阵芯片、原位杂交、RNase保护测定等各种分子生物学实验组分：应用范围：核酸提取及纯化使用方法：一、实验准备： 1.所有试剂用DEPC处理过的溶剂配制。请选用RNase-free枪头和离心管，以避免提取过程中RNA被RNase降解。 2.70%乙醇,- 20C预冷。二、操作步骤：不同的样品提取miRNA的操作中略有区别，具体步骤分述如下：【从动物组织中提取miRNA】 1.取20-40 mg组织，转移至预冷的研钵中，加液氮研磨成粉末。请按下面说明使用组织的量： ①RNA丰富的组织(如肝脏)：不超过30 mg ②RNA含量低的组织(如肌肉)：不超过100 mg ③当使用的组织量小于20 mg时：R-I，R- II和异丙醇的使用量都减半 ④当使用的组织量大于40 mg时：R-I，R-II和异丙醇的使用量按比例增加 2.加入400 ul Buffer R- I，用装有21-25号针头的注射器反复抽吸8-10次，转入1.5 m\|离心管(试剂盒内提供)中。 3.加入150 μl BufferR-1l,漩涡振荡15-30 s，12,000X g离心5 min。【建议在4℃下离心】 4.取上清至1.5 ml离心管中，加入180 u无水乙醇，混和均匀。 5.将制备管置于2 m\|离心管(试剂盒内提供)中，转移步骤4中的混合液到制备管中，12,000X g离心1 min。【①建议在4℃下离心；②miRNA在滤液中，注意保存滤液。】 6.弃制备管，在滤液中加入500 μl异丙醇，混和均匀。 7.12,000X g离心10 min,弃上清。 8.加入700 μl 70%乙醇( - 20℃预冷)，12,000Xg离心5min. 9.弃上清,室温干燥5- 10 min。 10.离心管中加入70 ul Buffer TE (nucdease-free) 或RNase-free水洗脱miRNA。【从植物组织中提取miRNA】 1.取30-150 mg组织，转移至预冷的研钵中，加液氮研磨成粉末。请按下面说明使用组织的量： ①植物叶子：通常用量10-80 mg ②植物纤维组织：通常用量100-150 mg ③当植物叶组织量小于30 mg时：R-I，R-II和异丙醉的使用量都减半 ④当植物叶组织量大于80 mg时：R-I，R-II 和异丙醇的使用量按比例增加 ⑤当植物纤维组织量大于150 mg时：R-I，R- II和异丙醇的使用量按比例增加 2.加入400 ul BufferR- I，用装有21-25号针头的注射器反复抽吸8-10次，转入1.5 mI离心管( 试剂盒内提供)中。. 3.加入150 ul Buffer R-1I,漩涡振荡15-30 s, 12.000x g离心5 min。【建议在4℃下离心】 4.取上清至1.5 ml离心管中，加入180 山无水乙醇，混和均匀。 5.将制备管置于2 mI离心管(试剂盒内提供)中，转移步骤4中的混合液到制备管中，12.000xg离心1 min.【①建议在4℃下离心；②miRNA在滤液中，注意保存滤液。】 6.弃制备管，在滤液中加入500 μl异丙醇，混和均匀。 7. 12,000xg高心10 min,弃上清。 8.加入700 ul 70%乙醇( - 20℃预冷)，12,000xg离心5min. 9.弃上清,室温干燥5 -10 min, 10.离心管中加入70 μl Buffer TE (nuclease-free) 或RNase-free水洗脱MiRNA。【从细胞中提取miRNA】步骤1-3根据细胞培养的方式不同可以选择a或b两种实验方法 a.悬浮培养的动物细胞或从培养皿、培养瓶中获得的细胞悬浮液或新鲜分离的动物组织单细胞悬浮液: 1a.收集2X 10-1X 10’的细胞，2,000Xg离心5 min，弃上清； 2a.加入400 μl BufferR- I，用装有21-25号针头的注射器反复抽吸8-10次，转入1.5mI离心管(试剂盒内提供)中； 3a. 加入150 μl Buffer R1I，旋涡振荡15-30s，12.000X g离心5 min.【建设在4℃下离心】。 b. 96-孔L, 24-孔,12-孔或6-孔培养板上贴壁培养的细胞: 1b.从96-孔， 24-孔，12-孔或6-孔培养板里收集细胞，尽量弃去培养基，每孔加入400 u山l Buffer R-I，用移液枪上下吹打8-10次； 2b.转移上述细胞悬浮液到1.5 ml离心管( 试剂盒内提供)中，用装有21-25号针头的注射器反复抽吸8-10次； 3b. 加入150 μl Bufflr R-II，漩涡振荡15-30 s， 12,000X g离心5 min.【建议在4℃下离心】 4.取上清至1.5 ml离心管中，加入180 山无水乙醇，混和均匀。 5.将制备管置于2 ml离心管(试剂盒内提供)中，转移步骤4中的混合液到制备管中，12.000Xg离心1 min。【①建议在4℃下离心；②miRNA在滤液中，注意保存滤液。】 6.弃制备管，在滤液中加入500 u异丙醇，混和均匀。 7.12,000Xg高心10 min,弃上清。 8.加入700 μ 70%乙醇( - 20℃预冷)，12,000xg离心5min. 9.弃上清,室温干燥5 -10 min. 10.离心管中加入70 ul Bufer TE (nucdease-free )或RNase-free水洗脱mRNA。三、流程图注意事项： Buffer R- I含刺激性化合物，操作时要戴乳胶手套和眼镜，避免沾染皮肤、眼睛和衣服，谨防吸入口鼻。若沾染皮肤、眼睛时，要立即用大量清水或生理盐水冲洗，必要时寻求医疗咨询。 This kit is used to extract miRNAs from various animal tissues, plant tissues and cells. The extracted miRNA molecule is complete and high purity, which is suitable for various molecular biology experiments such as Northern blot, real timepcr, miRNA microarray chip, in situ hybridization, RNase protection assay, etc Composition: Scope of application：* Nucleic acid extraction and purification Instruction： 1.Experimental preparation： 1.1.All reagents were prepared with DEPC-treated solvents. Please use RNase-free tip and centrifuge tube to avoid RNA degradation by RNase during extraction. 1.2.70 % ethanol, -20C pre-cooling. 2.Operational procedure： There is a slight difference in the operation of miRNA extraction from different samples. The specific steps are as follows : 【 Extraction of miRNA from animal tissues】 1.Take 20-40 mg tissue, transfer to a pre-cooled mortar, and add liquid nitrogen to grind into powder. Please click below to describe the amount of organization used : ①RNA-rich tissue ( e.g. liver ) : no more than 30 mg ②Tissues with low RNA content ( e.g., muscle ) : no more than 100 mg ③When the amount of tissue used was less than 20 mg : the amount of R-I, R-II and isopropanol used was halved. ④When the amount of tissue used was more than 40 mg : the use of R-I, R-II and isopropanol increased proportionally. 2.Add 400 ul Buffer R-I, repeatedly aspirate 8-10 times with a syringe equipped with a 21-25 needle, and transfer to a 1.5 m \| centrifuge tube ( provided in the kit ). 3.Add 150 μl BufferR-1l, swirl for 15-30 s, centrifuge at 12,000 X g for 5 min. [ Centrifugation at 4 °C is recommended ] 4.Take the supernatant to 1.5ml centrifuge tube, add 180 u anhydrous ethanol, mix evenly. 5.The preparation tube was placed in a 2 m \| centrifuge tube ( provided in the kit ), the mixture in step 4 was transferred to the preparation tube, and 12,000 X g was centrifuged for 1 min. [ 1 Centrifugation at 4 °C is recommended ; 2 miRNA in the filtrate, pay attention to preserve the filtrate. ] 6.Abandon the preparation tube, add 500μl isopropanol to the filtrate, and mix evenly. 7.12,000Xg centrifuged for 10 min, discard the supernatant. 8.Add 700μl 70 % ethanol ( pre-cooled at -20 °C ), centrifuged at 12,000Xg for 5min. 9.The supernatant was discarded and dried at room temperature for 5-10 min. 10.70 ul Buffer TE ( nucdease-free ) or RNase-free water was added to the centrifuge tube to elute miRNA. 【 Extraction of miRNA from plant tissue 】 1.Take 30-150 mg tissue, transfer to a pre-cooled mortar, and add liquid nitrogen to grind into powder. Please click below to describe the amount of organization used : ①Plant leaves : usually 10-80 mg ② Plant fiber tissue : usually 100-150 mg ③When the amount of plant leaf tissue was less than 30 mg : the amount of R-I, R-II and isopropyl alcohol used was halved. ④When the amount of plant leaf tissue was more than 80 mg : the use of R-I, R-II and isopropanol increased proportionally. ⑤When the amount of plant fiber tissue was more than 150 mg : the use of R-I, R-II and isopropanol increased proportionally. 2.Add 400 ul BufferR-I, use a syringe with a 21-25 needle to repeatedly suck 8-10 times, and transfer to a 1.5mI centrifuge tube ( provided in the kit ).. 3.Add 150 ul Buffer R-1I, vortex oscillation 15-30 s, 12.000 x g centrifugation 5 min. [ Centrifugation at 4 °C is recommended ] 4.Take the supernatant to 1.5ml centrifuge tube, add 180 mountain anhydrous ethanol, mix evenly. The preparation tube was placed in a 2 mI centrifuge tube ( provided in the kit ), the mixture in step 4 was transferred to the preparation tube, and 12.000 xg was centrifuged for 1 min. It is recommended to centrifuge at 4 °C ; 2 miRNA in the filtrate, pay attention to preserve the filtrate. ] Abandon the preparation tube, add 500μl isopropanol to the filtrate, and mix evenly. 7.12,000xg high heart for 10 min, discard the supernatant. 8.Add 700 ul 70 % ethanol ( -20 °C precooling ), 12,000 xg centrifuge for 5 min. 9.The supernatant was discarded and dried at room temperature for 5-10 min. 10.70 ul Buffer TE ( nucdease-free ) or RNase-free water was added to the centrifuge tube to elute miRNA. 【miRNA extraction from cells】 Steps 1-3 According to the different ways of cell culture, two experimental methods, a or b, can be selected. a. Suspension cultured animal cells or cell suspension obtained from petri dishes or culture flasks or freshly isolated animal tissue single cell suspension : 1a.Collect 2X 10 * -1X 10 ' cells, centrifuge 2,000Xg for 5 min, discard the supernatant ; 2a. Add 400 μl Buffer R-I, repeatedly draw 8-10 times with a syringe containing 21-25 needles, and transfer to a 1.5 mI centrifuge tube ( provided in the kit ) ; 3a. Add 150μl Buffer R1I, vortex oscillation 15-30s, 12.000Xg centrifugal 5min. [ build at 4 °C centrifugal ]. b. Cells cultured on 96-well L, 24-well, 12-well or 6-well plates : Cells were collected from 96-well, 24-well, 12-well or 6-well culture plates, and the medium was discarded as much as possible, and 400 u / well Buffer R-I was added to each well, and the pipette gun was used to blow up and down 8-10 times ; 2b.Transfer the above cell suspension to a 1.5ml centrifuge tube ( provided in the kit ), and repeatedly draw 8-10 times with a syringe containing 21-25 needles ; 3b. Add 150 μl Bufflr R-II, swirl for 15-30 s, centrifuge for 5 min at 12,000 × g. [ Recommended at 4 °C ] 4.Take the supernatant to 1.5ml centrifuge tube, add 180 mountain anhydrous ethanol, mixing evenly. 5.The preparation tube was placed in a 2 ml centrifuge tube ( provided in the kit ), the mixture in step 4 was transferred to the preparation tube, and centrifuged at 12.000 Xg for 1 min. [ 1 Centrifugation at 4 °C is recommended ; 2 miRNA in the filtrate, pay attention to preserve the filtrate. ] 6.Abandon the preparation tube, add 500 u of isopropanol to the filtrate, and mix evenly. 7.12,000Xg high heart for 10 min, discard the supernatant. 8.Add 700μ70 % ethanol ( pre-cooled at − 20 °C ), centrifuged at 12,000 × g for 5 min. 9.Abandon the supernatant, dry at room temperature for 5 - 10 min. 10.70 ul Bufer TE ( nucdease-free ) or RNase-free water was added to the centrifuge tube to elute mRNA. 3.Flow chart Matters needing attention： Buffer R-I contains irritating compounds, when operating to wear latex gloves and glasses, to avoid contamination of the skin, eyes and clothes, be careful not to inhale the nose and mouth. If the skin, eyes, to immediately rinse with a lot of water or saline, if necessary, seek medical advice.

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批号(Lot Number)	证书类型	货号
L2325105	分析证书	M598102

溶液计算器

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稀释计算器

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miRNA小量制备试剂盒

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