PicoGreen dsDNA 定量检测试剂盒
基本信息
| 产品名称 | PicoGreen dsDNA 定量检测试剂盒 |
|---|---|
| 英文名称 | PicoGreen dsDNA Assay Kit |
| 运输条件 | 冰袋运输 |
一般描述
产品介绍
用于 dsDNA 定量检测的 PicoGreen荧光染料
1.应用说明
在分子生物学的试验过程中，PicoGreen dsDNA 定量试剂盒是荧光检测双链 DNA 并进行定量 的一种产品，这种方法非常灵敏。常用于分子生物学技术中的：cDNA 文库的构建；用于亚克隆的 DNA 片段纯化及应用，比如进行 DNA 定量、产物扩增和引物的进一步检测。 疫苗是现代疾病预防中常用的控制方式。如今许多疫苗是细胞培养疫苗，比如重组乙肝疫苗、狂犬病疫苗等大多数疫苗都采用细胞培养的方法生产。其中，疫苗的纯化是关键问题，我们需要尽可能的去除宿主细胞DNA和宿主蛋白。假若宿主细胞的DNA和蛋白同疫苗一起注入人体将会产生不可预料的后果。
常规的DNA含量的检测方法是在260nm（A260）处测其吸光值。这种方法的主要缺点是核苷酸、单链核酸和蛋白质对信号的影响很大，并且还会受到核酸制备过程中污染物的干扰，无法区分DNA和RNA，而且这种方法不灵敏（5μg/mLdsDNA溶液A260=0.1）。PicoGreen定量检测方法简单、方便，被多家生物制品厂所选择，成为生物制品残留DNA检测的标准。
目前该方法已纳入2010年版《中国药典》
原理:
PicoGreen与DNA双链结合后才发出的荧光，无DNA不发荧光；所发荧光与DNA浓度成正比。在2010年《中国药典》中提出，PicoGreen定量DNA的方法检出限约0.3ng/ml，DNA含量在1.25-80ng/mL范围时线性较好(R2>0.99).
优点：
1）该方法可以测定来源于任何表达宿主样品中的双链DNA。
2）可以直接定量PCR扩增产物而无需从反应混合物中纯化DNA。
3）远远超出传统紫外A260的检测方法和Hoechst33258的灵敏度。
4）较高浓度的盐，尿素，乙醇，氯仿，去垢剂，蛋白或琼脂糖对测定无影响。
5）在等摩尔浓度 ssDNA 和 RNA 存在的条件下测定 dsDNA，其影响很小。
2.所需器材
• 微型荧光计；便携式荧光仪-上海互帼科学仪器有限公司HG-9型；1cm石英比色皿
• PicoGreen dsDNA 定量检测试剂盒, 1mL 单位量的试剂浓缩液足够 2mL 体积的 200 次测定。
1×TE（10mM Tris 1mM EDTA）pH8.0； 250ug/mL Sigma 小牛胸腺DNA
3.实验方案
试剂制备
PicoGreen dsDNA 定量试剂是以 1mL 的浓缩液形式保存在无水的 DMSO（二甲基亚砜）中。实 验当天，配制 2XPicoGreen 试剂的操作溶液，用 1xTE 按 1:200 的比例稀释浓缩液（10mM Tris-HCl，1mM EDTA，pH7.5）。如果要准备足够的操作溶液测定 20 个样品，可在 20mL1x TE 中加入 100μL PicoGreen dsDNA 定量试剂。由于试剂容易吸附到玻璃表面，要在塑料容器中配制。PicoGreen 试剂 见光易降解，所以应将配好的溶液用箔包住或放置暗处避光保存。
溶液最好在配制好数小时内使用，以保证最佳结果。
实验方法：
1). 标准品工作液的配制：
Sigama小牛胸腺嘧啶DNA干粉（货号：D4522-1MG）1mg（Tris，Nacl等浓度已成标准体系），加入1mL双蒸水，配制成1mg∕mL的标准品工作液；
2). 染料工作液的配置：
6 uLPicoGreen加入1mL TE(注意：用1×TE将PicoGreen稀释200倍，现用现配，注意避光)。
3). 标准品工作液稀释：
（1）母液稀释：取10ul（1mg∕mL）标准品工作液加入到990ul TE溶液中，浓度稀释成10ug∕mL，取10ul（10ug∕mL）标准品工作液加入到990ul TE溶液中，浓度稀释成100ng∕mL；
（2）倍比稀释：取800ul （100ng∕mL）的标准品工作液加入到200ul TE溶液中，浓度达到80ng∕mL（药典规定：荧光染色方法DNA含量在1.25-80 ng/mL范围线性较好， 该法DNA检出限为0.3 ng/ml），取500ul（80ng∕mL）的标准品工作液加入到500ul TE溶液中，浓度稀释到40ng∕mL；依次倍比稀释，配成20ng/ml、10ng/ml、5.0ng/ml、2.5ng/ml、1.25ng/ml、0.625ng/ml的标准品溶液；
4).标准曲线的制备：倍比稀释后的各梯度标准品溶液和染料工作液各取100ul混匀，避光室温放置5min。使用FB-15型便携式荧光仪检测样品的荧光值：将混合后的溶液加入微量比色皿，确信不要在样品中引入气泡，轻轻地弹微量检测皿的外部，可以驱散气泡。以1×TE缓冲液为blank，测定样品和空白对照的荧光值；用标准品溶液的浓度（ng/ml）对应的荧光强度作直线回归，制备标准曲线。
5). 测量剩余样品的荧光值。荧光计将给出一个直接的浓度读数，数据可以用来产生DNA 浓度的标准曲线。
产品参数
Ex(nm) 488 Em(nm) 520
Product description
PicoGreen fluorescent dye for dsDNA quantitative detection
1. Application description
In molecular biology experiments, the PicoGreen dsDNA quantification kit is a product for fluorescent detection and quantification of double-stranded DNA. This method is very sensitive. Commonly used in molecular biology technology: cDNA library construction; DNA fragment purification and application for subcloning, such as DNA quantification, product amplification, and further primer detection. Vaccines are a commonly used control method in modern disease prevention. Many vaccines today are cell culture vaccines, such as recombinant hepatitis B vaccine, rabies vaccine and most of the vaccines are produced using cell culture methods. Among them, the purification of vaccines is a key issue, and we need to remove host cell DNA and host proteins as much as possible. If the host cell’s DNA and protein are injected into the human body together with the vaccine, it will have unpredictable consequences.
The conventional DNA content detection method is to measure its absorbance at 260nm (A260). The main disadvantage of this method is that nucleotides, single-stranded nucleic acids and proteins have a great influence on the signal, and are also interfered by contaminants in the nucleic acid preparation process, and cannot distinguish between DNA and RNA, and this method is not sensitive (5μg) /mLdsDNA solution A260=0.1). The PicoGreen quantitative detection method is simple and convenient, and has been selected by many biological product manufacturers, and has become the standard for the detection of residual DNA in biological products. At present, this method has been included in the 2010 edition of “Chinese Pharmacopoeia”
Principle:
The fluorescence emitted after PicoGreen is combined with the double strand of DNA, without DNA, does not emit fluorescence; the fluorescence emitted is proportional to the concentration of DNA. It was proposed in the Chinese Pharmacopoeia in 2010 that the detection limit of PicoGreen method for quantifying DNA is about 0.3ng/ml, and the linearity is better when the DNA content is in the range of 1.25-80ng/mL (R2>0.99).
Advantage:
1) This method can measure double-stranded DNA in samples derived from any expression host.
2) It is possible to directly quantify PCR products without purifying DNA from the reaction mixture.
3) Far beyond the traditional UV A260 detection method and the sensitivity of Hoechst33258.
4) Higher concentrations of salt, urea, ethanol, chloroform, detergent, protein or agarose have no effect on the determination.
5) Measuring dsDNA in the presence of equimolar concentrations of ssDNA and RNA has little effect.
2. Required equipment
• Mini Fluorometer; Portable Fluorometer-Shanghai Hubei Scientific Instrument Co., Ltd. HG-9; 1cm quartz cuvette
• PicoGreen dsDNA Quantitative Detection Kit, a 1mL unit volume of reagent concentrate is sufficient for 200 determinations in a volume of 2mL.
1×TE (10mM Tris 1mM EDTA) pH8.0; 250ug/mL Sigma calf thymus DNA
3. Experimental protocol
Reagent preparation
PicoGreen dsDNA quantification reagent is stored in anhydrous DMSO (dimethyl sulfoxide) in the form of a 1mL concentrated solution. On the day of the experiment, prepare the operating solution of 2XPicoGreen reagent and dilute the concentrated solution (10mM Tris-HCl, 1mM EDTA, pH7.5) with 1xTE at a ratio of 1:200. If you want to prepare enough operating solution to measure 20 samples, you can add 100μL PicoGreen dsDNA quantification reagent to 20mL1x TE. Since the reagent is easily adsorbed to the glass surface, it should be prepared in a plastic container. PicoGreen reagent is easily degraded when exposed to light, so the prepared solution should be wrapped in foil or stored in a dark place away from light.
The solution is best used within a few hours of preparation to ensure the best results.
Experimental method:
1). Preparation of standard working solution:
Sigama calf thymine DNA dry powder (article number: D4522-1MG) 1mg (Tris, Nacl and other concentrations have become standard systems), add 1mL of double distilled water to prepare 1mg/mL standard working solution;
2). Dye working solution configuration:
6 Add 1mL TE to uLPicoGreen (Note: Dilute PicoGreen 200 times with 1×TE, and use it now, please avoid light).
3). Standard working solution dilution:
(1) Mother liquor dilution: Take 10ul (1mg/mL) standard working solution and add it to 990ul TE solution, dilute to 10ug/mL, take 10ul (10ug/mL) standard working solution and add it to 990ul TE solution, concentration Dilute to 100ng∕mL;
(2) Multiple dilution: Take 800ul (100ng/mL) standard working solution and add it to 200ul TE solution, the concentration reaches 80ng/mL (pharmacopoeia stipulates: the DNA content of fluorescent staining method is linear in the range of 1.25-80 ng/mL Ok, the DNA detection limit of this method is 0.3 ng/ml). Take 500ul (80ng/mL) of standard working solution and add it to 500ul TE solution, and dilute to 40ng/mL; successively dilute by multiples and make 20ng/mL. Standard solution of ml, 10ng/ml, 5.0ng/ml, 2.5ng/ml, 1.25ng/ml, 0.625ng/ml;
4). Preparation of standard curve: take 100ul of each gradient standard solution and dye working solution diluted by multiple ratios and mix them evenly, and place them at room temperature in the dark for 5 minutes. Use the FB-15 portable fluorometer to detect the fluorescence value of the sample: add the mixed solution into the micro cuvette, make sure not to introduce air bubbles into the sample, and gently flick the outside of the micro detection cuvette to disperse the air bubbles. Use 1×TE buffer as blank to measure the fluorescence values of the sample and the blank control; use the fluorescence intensity corresponding to the concentration of the standard solution (ng/ml) to make a linear regression to prepare a standard curve.
5). Measure the fluorescence value of the remaining samples. The fluorometer will give a direct concentration reading, and the data can be used to generate a standard curve of DNA concentration.
Product parameter
Ex(nm) 488 Em(nm) 520
相关属性
| 储存温度 | 2-8°C储存,避光,干燥 |
|---|---|
| 品牌 | Jinpan |