计算溶液所需的质量、体积或浓度。
为什么选择此级别
，适用于对基线干扰要求严格的色谱和分析工作流程。
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文献证明
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概述
产品内容
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* 本试剂盒适用于人基因组DNA文库构建，起始模板DNA投入量为5 ng。本公司还有针 对50 ng和1 ng的人基因组DNA起始的转座酶法文库构建试 剂盒，为得到质量较高的文库，不同的DNA起始量建议使用不同的试剂盒。
产品简介
该试剂盒是针对Illumina高通量测序平台定向开发的专用试剂盒，提供了基因组 DNA文库构建所需要的酶预混体系及反应缓冲液，包含除PCR引物外的所有成分。和 传统文库构建试剂盒相比，该试剂盒采用新型转座酶法进行文库构建，可以将DNA片 段化、末端修复及接头连接反应通过一步简单的酶促反应完成，显著降低了模板的使 用量，减少了实验操作步骤，缩短了文库构建时间；采用高保真DNA聚合酶进行文库 富集，无偏好的PCR扩增，扩大了序列的覆盖区域，可高效制备用于Illumina二代测序 平台的DNA文库。该试剂盒适用于起始模板DNA投入量为5 ng。试剂盒中所有试剂都 经过严格的质量控制和功能验证，最大程度上保证了文库构建的稳定性和重复性。
产品特点
● DNA片段化、接头连接一步完成。
● 超保真扩增，最大程度上降低了扩增偏好性。
自备仪器试剂盒耗材
1.磁力架：建议使用DynaMagTM-2。
2.DNA纯化回收试剂盒：建议使用磁珠法DNA纯化回收试剂盒。
3.文库PCR引物试剂盒：建议使用转座酶法二代测序多样本引物试剂盒。
4.无水乙醇，去离子水（pH 在7.0-8.0之间）。
5.反应管：建议使用低吸附的PCR管与1.5 ml离心管。 枪头：建议使用高质量过滤枪头防止试剂盒、文库样本污染。
实验前准备及重要注意事项
1.避免试剂的反复冻融。
2.PCR产物因操作不当极易产生污染，导致实验结果不准确，建议将PCR反应体系 配制区与PCR产物纯化区隔离，并使用专门的移液器，定时对各实验区域进行清 洁。
3.磁珠纯化：磁珠使用前应平衡至室温，磁珠的所有操作都应在室温下进行，80% 乙醇应现配现用，磁珠漂洗后干燥至表面无液体反光且呈磨砂状，磁珠干燥不足 会有乙醇残留影响后续实验，磁珠过度干燥影响DNA回收效率。
4.该试剂盒适合人基因组DNA文库构建，如DNA样品为PCR产物，应保证其长度＞ 500 bp，由于转座酶无法作用于DNA末端，推荐在制备PCR产物时将PCR产物两 末端各延长50-100 bp，以避免出现末端测序覆盖度低的情况。
样品准备
DNA纯度要求：A260/A280 = 1.8-2.0。 样本DNA：溶解于超纯水中。 DNA定量：DNA投入量过多、过少都会对文库质量有影响，建议先使用Nano检测基因 组DNA纯度再用Qubit对基因组进行浓度检测（请勿使用任何基于吸光度测量为基础的 测定方法进行模板定量）。
DNA建库流程示意图
操作步骤
DNA片段化、接头连接反应
1. 向200 μl PCR管中加入以下试剂：
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2.用移液器轻轻吹打混匀，短暂离心,使所有组分收集到管底。
3.将上述PCR管置于PCR仪中，热盖打开， 反应程序如下：
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失活反应
DNA在进行片段化反应完成后，酶仍处于较高的活性状态，应立即将其从PCR仪上取 下，加入反应终止Buffer进行终止反应，以防止DNA过度片段化导致的文库片段变小。 失活反应
1.向盛有片段化产物的PCR管中加入3 μl TS Buffer。
2.用移液器轻轻吹打混匀，短暂离心,使所有组分收集到管底。
3.置于室温孵育5 min，如室内温度过低，可置于PCR仪上进行反应，25℃，关闭热盖。
PCR扩增
1. 向200 μl PCR管中加入以下试剂：
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2.用移液器轻轻吹打混匀，短暂离心,使所有组分收集到管底。
3.将上述PCR管置于PCR仪中，热盖打开， 反应程序如下：
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文库DNA片段选择性回收
建议使用磁珠法DNA纯化回收试剂盒进行DNA片段选 择性回收。需要不同大小DNA片段时，磁珠使用量不同，具体磁珠使用量参照附表 （若使用其他品牌磁珠，需自行摸索最佳磁珠用量）。
注意：扩增产物也可使用胶回收试剂盒进行片段长度分选和纯化。如对文库长度分布范围无特殊要求， 扩增产物也可不进行DNA片段选择性回收，参考说明书第4页直接进行DNA片段的纯化。
1.CMPure使用前应震荡混匀后置于室温平衡30 min。
2.PCR产物转移至1.5 ml离心管中，补水至100 μl，加入若干体积平衡至室温的磁 珠，涡旋震荡5秒钟后，室温静置5分钟。
3.短暂离心，将离心管置于磁力架上，使磁珠和上清分离，直至溶液澄清，小心吸 取上清转移至一新的1.5 ml离心管中。 注意：不要弃除上清。
4.在上清中加入若干体积的磁珠，涡旋震荡5秒钟后，室温静置5分钟。
5.短暂离心，将离心管置于磁力架上，使磁珠和上清分离，直至溶液澄清，小心吸 取上清并弃除，期间避免接触已结合目标DNA的磁珠。 注意：不要弃除磁珠。
6.继续保持离心管固定于磁力架上，向离心管中加入200 μl新鲜配置的80%乙醇，室 温放置30秒，小心弃除上清。 注意：加乙醇时，液体不可直接吹打到磁珠上。
7.重复步骤6一次。
8.保持离心管固定于磁力架上，室温静置干燥至磁珠表面微裂，加入20 μl ddH2 O回溶。 注意：不要使磁珠过度干燥，以免影响洗脱效率。
9.将离心管从磁力架上取下，涡旋振荡使磁珠完全重悬，室温静置5分钟。短暂离 心，将离心管放于磁力架上直至溶液澄清，将上清溶液转移至一个新的离心管中。
附表：不同片段选择回收时磁珠建议用量
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文库DNA片段纯化
1.建议使用磁珠法DNA纯化回收试剂盒。
CMPure使用前应震荡混匀后置于室温平衡30 min。
2.PCR产物中加入50 μl平衡至室温的磁珠，涡旋震荡5秒钟后，室温静置5分钟。
3.短暂离心，将离心管置于磁力架上，使磁珠和上清溶液分离，直至溶液澄清（约 需3-5分钟），小心吸取上清并弃除，期间避免接触已结合目标DNA的磁珠。 注意：不要弃除磁珠。
4.继续保持离心管固定于磁力架上，向离心管中加入200 μl新鲜配置的80%乙醇，室 温放置30秒，小心弃除上清。 注意：加乙醇时，液体不可直接吹打到磁珠上。
5.重复步骤4。
6.保持离心管固定于磁力架上，室温静置干燥至磁珠表面微裂，加入25 μl ddH2 O回溶。 注意：不要使磁珠过度干燥，以免影响洗脱效率。
7.将离心管从磁力架上取下，涡旋振荡使磁珠完全重悬，室温静置5分钟。短暂离 心，将离心管放于磁力架上直至溶液澄清，将上清溶液转移至一个新的离心管中。
文库质量检测
文库浓度测定
为了得到高质量的测序结果，需要对DNA文库进行精确定量，首先推荐使用Real-time PCR方法对DNA文库进行绝对定量。此外，还可使用荧光染料法，如Qubit法或荧光染 料picogreen法，此处请勿使用基于吸光度测量的定量方法。最终可使用以下近似公式 换算DNA文库的摩尔浓度。
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文库片段分布
Products content
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* This kit is suitable for human genomic DNA library construction, the starting template DNA input is 5 ng. We also have transposase library construction kits for 50 ng and 1 ng of human genomic DNA starting, in order to get a higher quality library, it is recommended to use different kits for different starting amount of DNA.
Products Introduction
This kit is developed for Illumina's high-throughput sequencing platform and provides the enzyme premix system and reaction buffer for genomic DNA library construction, including all components except PCR primers. Compared with the traditional library construction kits, this kit adopts the new transposase method for library construction, which can complete DNA fragmentation, end repair and junction reaction in one simple enzymatic reaction, significantly reducing the amount of template, reducing the number of experimental steps, and shortening the time of library construction; it adopts the high-fidelity DNA polymerase for library enrichment, and the preference-free PCR amplification can expand the coverage area of the sequence, which can be used for efficient and effective sequencing. The use of high-fidelity DNA polymerase for library enrichment and preference-free PCR amplification broadens the coverage area of the sequence and enables efficient preparation of DNA libraries for Illumina's second-generation sequencing platform. The kit is suitable for use with a starting template DNA input of 5 ng, and all reagents in the kit have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction.
Product Features
● DNA fragmentation and junction ligation in one step.
● Ultra-fidelity amplification minimizes amplification preference.
Provide your own instruments, kits and consumables
1. Magnetic frame: DynaMagTM-2 is recommended.
2. DNA purification and recovery kit: It is recommended to use DNA purification and recovery kit by magnetic bead method.
3. Library PCR primer kit: It is recommended to use transposase method for second generation sequencing multi-sample primer kit.
4. Anhydrous ethanol, deionized water (pH between 7.0 and 8.0).
5. Reaction tubes: It is recommended to use low adsorption PCR tubes and 1.5 ml centrifuge tubes. Tips: It is recommended to use high quality filter tips to prevent contamination of kits and library samples.
Pre-experiment Preparation and Important Notes
1. Avoid repeated freezing and thawing of reagents.
2. PCR products are easily contaminated due to improper operation, resulting in inaccurate results. It is recommended to isolate the PCR reaction system preparation area from the PCR product purification area, and to use special pipettes to clean the experimental areas at regular intervals.
3. Bead purification: the beads should be equilibrated to room temperature before use, all operations on the beads should be carried out at room temperature, 80% ethanol should be dispensed freshly, the beads should be rinsed and dried until the surface is free of liquid reflections and has a frosted appearance, insufficient drying of the beads will cause ethanol residue that will affect the subsequent experiments, and over-drying of the beads will affect the efficiency of DNA recovery.
4. The kit is suitable for human genomic DNA library construction, if the DNA sample is a PCR product, it should be ensured that its length>.
500 bp, since transposases do not work on DNA ends, it is recommended to extend the PCR product by 50-100 bp at each end of the PCR product to avoid low coverage of the ends for sequencing.
Sample Preparation
DNA purity requirements: A260/A280 = 1.8-2.0. Sample DNA: dissolve in ultrapure water. DNA Quantification: Too much or too little DNA will affect the quality of the library. It is recommended to use Nano to test the purity of the genomic DNA and then use Qubit to test the concentration of the genome (do not use any absorbance-based assay for template quantification).
Schematic diagram of DNA banking process
procedure
DNA fragmentation, junction reaction
1. Add the following reagents to a 200 μl PCR tube:
2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.
3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows:
inactivation reaction
After the DNA is fragmented, the enzyme is still in a high active state, so it should be removed from the PCR instrument immediately and terminated by adding the Reaction Termination Buffer, in order to prevent the DNA from being fragmented too much and resulting in smaller library fragments.
1. Add 3 μl of TS Buffer to the PCR tube containing the fragmentation product.
2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.
3. Incubate at room temperature for 5 min, or if the room temperature is too low, place the reaction on a PCR instrument at 25°C with the thermal cover closed.
1. Add the following reagents to a 200 μl PCR tube.
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2. Mix by gently blowing with a pipette and centrifuge briefly so that all components are collected at the bottom of the tube.
3. Place the above PCR tubes in the PCR instrument with the hot cap on and program the reaction as follows
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Selective recovery of library DNA fragments
It is recommended to use CombiVision Magnetic Beads DNA Purification and Recovery Kit for selective recovery of DNA fragments. When different sizes of DNA fragments are required, the amount of magnetic beads used is different, please refer to the attached table for the specific amount of magnetic beads used.
(If using other brands of magnetic beads, you need to figure out the optimal amount of magnetic beads by yourself).
Note: Amplification products can also be fragment length sorted and purified using the Gum Recovery Kit. If there is no special requirement for library length distribution, amplification products can also be purified directly from DNA fragments without selective recovery of DNA fragments as described on page 4 of the manual.
1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.
2. Transfer the PCR products to a 1.5 ml centrifuge tube, rehydrate to 100 μl, add several volumes of magnetic beads equilibrated to room temperature, vortex for 5 seconds, and let stand at room temperature for 5 minutes.
3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, and carefully aspirate the supernatant and transfer it to a new 1.5 ml centrifuge tube.
Note: Do not discard the top clear.
4. Add several volumes of magnetic beads to the supernatant, vortex and shake for 5 seconds, then let stand at room temperature for 5 minutes.
5. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant until the solution is clear, carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA.
Note: Do not discard the beads.
6. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 μl of freshly prepared 80% ethanol to the tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.
Note: When adding ethanol, the liquid must not be blown directly onto the beads.
7. Repeat step 6 once.
8. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 20 μl of ddH2O to solubilize.
Note: Do not over-dry the beads as this may affect the elution efficiency.
9. Remove the centrifuge tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new centrifuge tube.
Table: Suggested amount of magnetic beads for different segment selection recovery
Library DNA fragment purification
We recommend the use of the Century Magnetic Bead Method DNA Purification and Recovery Kit.
1. CMPure should be equilibrated at room temperature for 30 min after shaking and mixing before use.
2. 50 μl of magnetic beads equilibrated to room temperature were added to the PCR product, vortexed and shaken for 5 seconds, and then left to stand at room temperature for 5 minutes.
3. Centrifuge briefly, place the tube on a magnetic rack to separate the beads from the supernatant solution until the solution is clear (approximately 3-5 minutes), carefully aspirate the supernatant and discard it, avoiding contact with the beads that have bound the target DNA. Note: Do not discard the beads.
4. Continue to keep the centrifuge tube fixed on a magnetic rack and add 200 μl of freshly prepared 80% ethanol to the centrifuge tube and allow to stand at room temperature for 30 seconds, carefully discarding the supernatant.
Note: When adding ethanol, the liquid must not be blown directly onto the beads.
5. Repeat step 4.
6. Keep the centrifuge tube fixed on a magnetic rack and leave to dry at room temperature until the surface of the beads is slightly cracked, add 25 μl of ddH O to solubilize.
Note: Do not over-dry the beads as this may affect the elution efficiency.
7. Remove the tube from the magnetic rack, vortex to completely resuspend the beads, and allow to stand at room temperature for 5 minutes. Centrifuge briefly, place the tube on the magnetic rack until the solution is clear, and transfer the supernatant solution to a new tube.
Library quality control
Determination of library concentration
In order to obtain high-quality sequencing results, accurate quantification of DNA libraries is required, and the first recommendation is to use Real-time
PCR methods are used for absolute quantification of DNA libraries. Additionally, fluorescent dye methods such as the Qubit method or the fluorescent dye picogreen method can be used; do not use quantification methods based on absorbance measurements here. The following approximate formula can be used to convert the molar concentration of the DNA library.
Library fragment distribution
The prepared DNA libraries can be detected by agarose gel electrophoresis or Agilent 2100 Bioanalyzer.
Range of segment length distributions.
规格
英文名称
NGS TPM DNA Library Prep Set for Illumina (5 ng)
储存与运输
储存条件
-20°C储存,避免反复冻融
运输条件
超低温运输
技术文档
高级数据
质检证书（CoA,COO,BSE/TSE 和分析图谱)
C of A & Other Certificates(BSE/TSE, COO):
输入批号以搜索分析图谱:
技术文档和文章
溶液计算器
常见问题
本产品应如何储存？
请于?20 °C条件下保存。避免反复冻融。建议按单次用量分装保存。
本产品如何运输？
本产品采用保温箱配冰袋运输。收货后请立即拆包，并按上述储存条件保存。
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