产品简介
该产品是从293T细胞中提取的高纯度的基因组DNA提取物，琼脂糖凝胶（0.7%） 电泳检测显示该DNA提取物大小在15Kb以上，且基本无降解，产品最终保存在TE Buffer中，可广泛应用于PCR、酶切、杂交、微阵列分析等分子生物学实验。
产品采用NanoDrop One定量浓度为200 ng/μL。
使用前准备及注意事项
建议-20˚C低温长期保存，使用前应先从冰箱内取出，平衡至室温后离心再打开瓶盖使用。样品开封后，应尽快恢复密封状态。
使用方法(以qPCR实验为例)
1. 扩增模板准备
将待检测样品用TE（10 mM Tris-Cl，pH8.0，1mM EDTA）稀释，稀释后浓度尽量在0.05-10 ng/μL之间。4℃冰上放置备用。
2. 标准品稀释：按照下表，先将Human DNA Standard 1（100ng/uL）用TE按下表稀释出5个不同浓度的标准品。10 ng/μL的DNA Standard 1（Std. 1）可在-20℃稳定保存1个月；Std2-5只能当天使用，配好后暂时不用时应4℃或冰上放置。
3. qPCR反应体系配制
配制前预先将所需要用到的冷冻保存的试剂完全融化并多次颠倒混匀，然后短暂离心后备用。20 μL的基础反应体系如下。
20 μL的基础反应体系如下：
注意： High Rox机型：每50 μL反应体系加入1 μL 50×High Rox； Low Rox机型：每500 μL反应体系加入1 μL 50×High Rox。
通常引物浓度以0.2 μM可以得到较好结果，可以在0.1-1.0 μM作为设定范围的参考。
使用的探针浓度，与使用的荧光定量PCR仪、探针种类、荧光标记物质种类有关，实际使用时请参照仪器说明书，或各荧光探针的具体使用要求进行浓度的调节。
根据需要配出足够量的反应体系混合物，反应体系配完并充分混匀后，按每孔16 μL 体积加入反应孔中。然后将准备好的标准品及稀释好的样品加入对应反应孔，加入量为4 μL/孔。空白对照管中加入TE，同样加入量为4 μL/孔。
推荐使用20 μL反应，如需进行更小体系反应，将体系各组分等比减少即可。
4. qPCR反应程序
以下举例为本公司GoldStar Probe Mixture产品反应条件为例，实际操作中应根据所用PCR 产品模板、引物结构和目的片段大小不同进行相应的改进和优化。
数据分析
1. 标准曲线制作
参照数据处理Excel表绘制标准曲线。标准曲线相关系数R2应不低于0.98，以Ct 值为纵坐标时，斜率应位于-3.1与-3.6之间，如标准曲线参数不合理，建议重复实验。
Products
This product is a high purity genomic DNA extract from 293T cells, agarose gel (0.7%) electrophoresis showed that the size of the DNA extract is more than 15Kb, and basically no degradation, the product is ultimately preserved in TE Buffer, which can be widely used in molecular biology experiments, such as PCR, enzyme digestion, hybridization, microarray analysis, and other molecular biology experiments.
The product was quantified using NanoDrop One at a concentration of 200 ng/μL.
Preparation and precautions before use
Long-term storage at -20˚C is recommended. Before use, the bottle should be removed from the refrigerator and equilibrated to room temperature and centrifuged before opening the cap for use. Samples should be restored to the sealed state as soon as possible after opening.
How to use (take qPCR experiment as an example)
1. Amplification template preparation
The samples to be detected were diluted with TE (10 mM Tris-Cl, pH 8.0,1 mM EDTA), and the concentration after dilution was as close as possible to the range of 0.05-10 ng/μL. The samples were placed on ice at 4°C and set aside.
2. Standard dilution: according to the following table, firstly dilute Human DNA Standard 1 (100ng/uL) with TE to make 5 different concentrations of standards according to the table below. 10ng/μL of DNA Standard 1 (Std. 1) can be stored stably at -20℃ for 1 month; Std2-5 can only be used on the same day, and should be placed at 4℃ or on ice when not in use for the time being after preparation. When not used temporarily after preparation, it should be stored at 4℃ or on ice.
style | Corresponding concentration (ng/μL) | Minimum dilution volume (in μL) | Std.1 | 10 | 10 [100 ng/μL DNA Standard 1] + 90 TE | Std.2 | 2.5 | 20 [Std. 1] +60 TE | Std.3 | 0.625 | 20 [Std. 2] +60 TE | Std.4 | 0.15625 | 20 [Std. 3] +60 TE | Std.5 | 0.0390625 | 20 [Std. 4] +60 TE |
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3. qPCR reaction system preparation
The cryopreserved reagents to be used were completely thawed and mixed by inversion several times before preparation, and then briefly centrifuged and prepared for use. 20 μL of the base reaction system was as follows.
The base reaction system for 20 μL was as follows:
reagents | 20μL reaction system | 2×qPCRMix | 10μL | PrimerMix | XμL | ProbeMix | XμL | Template | 4μL | ddH2O | Make up to 20 μL |
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Note: High Rox model: add 1 μL of 50×High Rox per 50 μL of reaction system; Low Rox model: add 1 μL of 50×High Rox per 500 μL of reaction system.
Usually, better results can be obtained with a primer concentration of 0.2 μM, and 0.1-1.0 μM can be used as a reference for setting the range.
The concentration of the probe used is related to the fluorescent quantitative PCR instrument used, the type of probe, and the type of fluorescent labeling substance, so please refer to the manual of the instrument or the specific requirements for the use of each fluorescent probe for the adjustment of the concentration during actual use.
Prepare a sufficient amount of reaction system mixture as required. After the reaction system has been prepared and mixed thoroughly, add 16 μL per well to the reaction wells. Then add the prepared standard and diluted sample into the corresponding reaction wells, the volume of addition is 4μL/well. TE was added to the blank control tube, and the same amount of TE was added at 4 μL/well.
It is recommended to use 20 μL for the reaction, if you need to perform a smaller system reaction, reduce the system components in equal proportion.
4. qPCR reaction program
The following is an example of our GoldStar Probe Mixture reaction conditions, which should be improved and optimized according to the PCR product template, primer structure and target fragment size.
move | temp | timing | circulate | premutability | 95°C | 10min | 1 | denaturation | 95°C | 10sec | 55 | Annealing/Extension | 60°C | 30sec | 5 |
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Data analysis
1. Standard curve production
The standard curve was plotted with reference to the Excel sheet for data processing. The correlation coefficient R2 of the standard curve should not be lower than 0.98, and the slope should be between -3.1 and -3.6 when the Ct value is the vertical coordinate. If the parameters of the standard curve are unreasonable, it is recommended to repeat the experiment.
ZJ25F0825986
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