PicoGreen 是一种高灵敏度、高特异性的双链 DNA (dsDNA) 染料，它可以与 dsDNA 特异性结合并发出绿色荧光，其荧光强度与样品中 dsDNA 的量成正比，所以可以通过检测荧光值对样品中 dsDNA 进行定量。本染料检测 dsDNA 的灵敏度非常高，其检测下限可达 pg/mL 级别。此外，相较于传统的 DNA 定量的方法紫外吸收法，PicoGreen 染料特异性很高，几乎不会受到样品中单链 DNA (ssDNA)、蛋白质和盐类的干扰，大大增加了定量的准确性。本染料可以应用于多种分子生物学实验，如 NGS 文库的准确定量与均一化；qPCR 或芯片分析前 DNA 样本质控；病毒载体滴度测定或细胞基因组 DNA 提取量评估；环境微生物 DNA 或法医样本等微量核酸的定量等。
【产品优势】
(1) 超高的灵敏度：可检测低至 25pg/mL 的 dsDNA；
(2) 良好的特异性：对 RNA、单链 DNA 或游离核苷酸的反应极弱，背景干扰小；
(3) 高荧光信号：对 dsDNA 的荧光信号增强约 1000 倍；
(4) 宽广的线性范围：定量 dsDNA 可跨 4 个数量级；
(5) 操作简便快速：只需将染料加入样品中，孵育后即可检测；
(6) 安全环保：Picogreen 染料毒性低，使用很安全。
【使用方法】
实验前准备试剂: 1×TE Buffer、标准 DNA
一、使用 Qubit® 荧光仪进行 dsDNA 定量分析 以 10μL dsDNA 样品为例，
1. 将 PicoGreen 染料取出至室温下，待其完全溶解后，震荡混匀，短暂离心。
2. 用 1×TE Buffer 将染料稀释 200 倍，得到染料工作液；
3. 根据检测样品数量，向 Qubit 分析管中加入 190μL 工作液；
4. 向分析管中加入 dsDNA 标准品 1、dsDNA 标准品 2，或待测的 dsDNA 样本，每管 10μL，涡旋震荡 2-3 秒，室温下避光孵育 1min。
5. 按照 Qubit® 荧光仪的操作步骤，选择 dsDNA High Sensitivity 检测程序测定荧光信号值。
二、使用酶标仪进行 dsDNA 定量检测分析 以 10μL dsDNA 样品为例，
1. 将 PicoGreen 染料取出至室温下，待其完全溶解后，震荡混匀，短暂离心。
2. 用 1×TE Buffer 将染料稀释 200 倍，得到染料工作液；
3. 根据检测样品数量，向 96 孔酶标板中加入 190μL 工作液；
4. 将标准 DNA 稀释成 5 个不同梯度，待测样本 dsDNA 浓度应介于这 5 个浓度梯度范围内；
5. 向 96 孔酶标板加入待测 dsDNA 样品和标准浓度样本，室温孵育 2min;
6. 使用酶标板检测荧光信号值，设置激发波长为 480nm，发射波长设置为 520nm;
7. 根据标准品浓度和荧光信号值绘制标准曲线，然后根据标准曲线和各样品荧光值计算出待测样品浓度。
【注意事项】
1、为了您的安全和健康，请穿实验服并戴一次性手套操作；
2、本品仅适用于科研用途。
PicoGreen is a high-sensitivity and high-specificity double-stranded DNA (dsDNA) stain. It binds specifically to dsDNA and emits green fluorescence, and its fluorescence intensity is proportional to the amount of dsDNA in the sample. Therefore, dsDNA in samples can be quantified by measuring fluorescence readouts. This stain delivers ultrahigh sensitivity for dsDNA detection, with a limit of detection down to the pg/mL level. In addition, compared with the traditional UV absorbance method for DNA quantification, PicoGreen exhibits excellent specificity; it is barely interfered by single-stranded DNA (ssDNA), proteins and salts in samples, which greatly improves quantification accuracy. This stain is applicable to a wide range of molecular biology experiments, including accurate quantification and normalization of NGS libraries; DNA sample quality control prior to qPCR or microarray analysis; viral vector titer determination and evaluation of cellular genomic DNA extraction yield; quantification of trace nucleic acids such as environmental microbial DNA and forensic samples.
Product Advantages
1. Ultrahigh sensitivity: Detects dsDNA at concentrations as low as 25 pg/mL;
2. Excellent specificity: Shows negligible response to RNA, single-stranded DNA or free nucleotides, resulting in low background interference;
3. Strong fluorescence signal: Fluorescence signal enhancement of approximately 1,000-fold upon binding to dsDNA;
4. Wide linear dynamic range: Quantifies dsDNA across 4 orders of magnitude;
5. Simple and rapid workflow: Simply add the stain to samples and incubate before detection;
6. Safe and eco-friendly: PicoGreen has low toxicity and ensures safe handling.
Operating Procedures
Reagents Required Before Experiment: 1× TE Buffer, Standard DNA
I. dsDNA Quantification with Qubit® Fluorometer
Take a 10 μL dsDNA sample as an example:
1. Equilibrate PicoGreen stain to room temperature, fully dissolve it, vortex thoroughly and perform a brief centrifugation.
2. Dilute the stain 200-fold with 1× TE Buffer to prepare working stain solution.
3. Add 190 μL working solution into Qubit assay tubes according to the number of test samples.
4. Add 10 μL of dsDNA Standard 1, dsDNA Standard 2 or unknown dsDNA sample to each tube, vortex for 2–3 seconds, and incubate at room temperature in the dark for 1 min.
5. Follow the operating instructions of the Qubit® fluorometer, select the dsDNA High Sensitivity assay program to measure fluorescence signal values.
II. dsDNA Quantification with Microplate Reader
Take a 10 μL dsDNA sample as an example:
1. Equilibrate PicoGreen stain to room temperature, fully dissolve it, vortex thoroughly and perform a brief centrifugation.
2. Dilute the stain 200-fold with 1× TE Buffer to prepare working stain solution.
3. Add 190 μL working solution into a 96-well microplate based on the number of samples to be tested.
4. Prepare standard DNA with 5 distinct concentration gradients; the dsDNA concentration of unknown samples should fall within the range of these 5 gradients.
5. Load unknown dsDNA samples and standard concentration samples into the 96-well microplate, then incubate at room temperature for 2 min.
6. Measure fluorescence signals via the microplate reader, set excitation wavelength to 480 nm and emission wavelength to 520 nm.
7. Generate a standard curve based on standard concentrations and corresponding fluorescence signals, then calculate the concentration of unknown samples using the standard curve and sample fluorescence values.
Precautions
1. Wear a lab coat and disposable gloves during operation for personal safety and health protection.
2. This product is for research use only.
ZJ26F0939358
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