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。
储存与运输
-20°C储存,避免反复冻融。超低温运输 。请查阅批次 COA 获取详细规格。
质量文档
SDS、COA、产品数据表及规格说明书均可下载。可通过批号查询获取批次 COA。
文献证明
在色谱分析、有机合成和交叉偶联反应领域已被 0 篇同行评审文献引用。
概述
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产品简介
二代测序快速DNA建库试剂盒(Ion torrent)提供了构建DNA文库需要的酶预混体系及反应缓冲液，包含除接头以外的所有成分，制备的文库可用于Ion torrent PGM、Ion Proton二代测序平台的测序。和常规建库方法相比，该试剂盒将多个步骤进行了合并， 省略了多个纯化步骤，因此显著降低了起始模板DNA的最少需求量，缩短了文库构建时间。另外，试剂盒采用高保真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，可高效的制备用于Ion torrent二代测序平台的DNA文库。
自备仪器、试剂和耗材
1．磁力架。2．DNA纯化回收试剂盒。
3．样本接头引物试剂盒。
4．无水乙醇，EB (10 mMTris-HCl, pH 8.0)，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．避免试剂盒中Buffer的反复冻融，建议首次使用时将Buffer进行分装保存。酶在使 用完应尽快放回-20℃保存，
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，定期对各实验区域进行清洁。
DNA建库流程示意图：
起始材料：5 ng-1 μg打断的双链DNA，溶于EB（10 mM Tris-HCl pH 8.0）或去离子水中， DNA纯度要求：OD260/OD 280=1.8-2.0。
DNA末端修复反应：
1．向200 μl PCR管中加入以下成分，用枪头将上述溶液轻轻混匀，瞬时离心使所有组
分收集到管底。
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2．将管子置入PCR仪中，热盖打开，反应程序如下：
20 min @ 25℃
10 min @ 70℃
Hold on 4℃
Adaptor 连接：
以下为使用adaptor进行连接的操作步骤：
1．向上述反应液中直接加入以下试剂, 用枪头将上述试剂混匀，短暂离心，使溶液收集到管底。
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注：建议Adaptor的加入量与DNA片段的摩尔比为10:1-20:1，具体Adaptor的使用浓度请参照下表。如 果DNA量为10-100 ng, adaptor建议使用浓度为1 μM（小于260 bp）或 0.5μM（300-400 bp）。
2．反应步骤
15 min @ 25℃
5 min @ 65℃
Hold on 4℃
Adaptor连接DNA片段的选择性回收
构建不同大小的DNA文库时，需进行DNA片段的选择性回收。若起始样本量低于 50ng，不建议进行DNA片段选择性回收。可参考另一种方案，直接进行DNA片段的纯 化。以下操作步骤采用磁珠法DNA纯化回收试剂盒，可选择回收DNA片段长度范围为310-370bp（读 长为200bp），反应起始体积为100 μl。
1．涡旋振荡CMPure20秒，使其彻底混匀为均一溶液；
2．将100μl adaptor连接反应缓冲液转移至一新的1.5ml离心管中；
3．加入60 µl混合均匀的CMPure，涡旋震荡5秒钟后室温静置5分钟；
4．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5 分钟），小心地将上清溶液转移至新的1.5ml离心管中,并弃去磁珠；
注意：不要弃除上清。
5．向上清中加入20µl混合均匀的CMPure，涡旋震荡5秒钟后室温放置5分钟；
6．短暂离心，将离心管放于磁力架上，使磁珠和上清溶液分离，直至溶液澄清（约需5 分钟），小心移取上清并弃除，期间避免接触结合目标DNA的磁珠；
注意：不要弃除磁珠。
7．继续保持离心管固定于磁力架上，向离心管中加入250 µl新配置的80%乙醇，室温放置30秒，待悬起的磁珠完全吸附后，小心弃除上清；
8．重复步骤7；为彻底移除残留液体，可将离心管短暂离心后，再次移除残留液体。
9．保持离心管固定于磁力架上，室温静置5分钟，使磁珠在空气中干燥；
10.将离心管从磁力架上取下，加入25 µl 10 mMTris-HCl（pH 8.0）或去离子水（自备）， 涡旋振荡使磁珠完全重悬于洗脱液中，室温静置5分钟；
11.短暂离心，将离心管放于磁力架上直至溶液澄清（约需5分钟），将25 μl洗脱液转移 至一个新的PCR管中；
另一种方案：Adaptor连接DNA片段的全部回收
1．涡旋振荡CMPure20秒，使其彻底混匀为均一溶液。
2．将adaptor连接反应液转移至一新的1.5 ml离心管中。
3．加入1倍样品体积的CMPure，涡旋震荡5秒钟后，室温静置5分钟。
4．短暂离心，将离心管放于磁力架上，使磁珠和上清溶液分离，直至溶液澄清（约需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。
5．继续保持离心管固定于磁力架上，向离心管中加入250 μl新鲜配置的80%乙醇，室温 放置30秒，待悬起的磁珠完全吸附后，小心弃除上清。
6．重复步骤5。为彻底移除残留液体，可将离心管短暂离心后，再次移除残留液体。
7．保持离心管固定于磁力架上，室温静置5分钟，使磁珠在空气中干燥。
8．将离心管从磁力架上取下，加入25 μl EB（自备）或去离子水，涡旋振荡使磁珠完全 重悬于洗脱液中，室温静置5分钟。
9．短暂离心，将离心管放于磁力架上直至溶液澄清（约需5分钟），将25 μl洗脱液转移
至一个新的PCR管中。
PCR富集
1．在PCR管中加入以下试剂并混匀
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2．PCR反应条件
注：样本量为1ug时，4-6个cycles，样本量100ng时6-8个cycles，样本量为10ng时10-12个cycles。
PCR循环数可根据实验进行优化。
PCR产物的纯化
1．涡旋振荡CMPure20秒，使其彻底混匀为均一溶液；
2．将PCR反应液转移至一新的1.5 ml离心管中；
3．加入1倍样品体积的CMPure，涡旋震荡5秒钟后室温静置5分钟；
4．短暂离心，将离心管放于磁力架上，使磁珠和上清溶液分离，直至溶液澄清（约需5 分钟）。小心移取上清并弃除，期间避免接触结合目标DNA的磁珠； 注意：不要弃除磁珠
5．继续保持离心管固定于磁力架上，向离心管中加入250 µl新鲜配置的80%乙醇，室温 放置30秒，待悬起的磁珠完全吸附后，小心弃除上清。
6．重复步骤5；为彻底移除残留液体，可将离心管短暂离心后，再次移除残留液体。
7．保持离心管固定于磁力架上，室温静置5分钟，使磁珠在空气中干燥。
8．将离心管从磁力架上取下，加入25 µl EB（自备）或去离子水，涡旋振荡使磁珠完全 重悬于洗脱液中，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 （约需5分钟），将25 μl洗脱液转移至一个新的PCR管中，DNA文库在-20℃保存。
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Product Introduction
Second Generation Sequencing Rapid DNA Library Construction Kit (Ion torrent) provides the enzyme premix system and reaction buffer required for constructing DNA libraries, including all components except junctions, and the prepared libraries can be used for sequencing on Ion torrent PGM and Ion Proton second generation sequencing platforms. Compared with conventional library construction methods, the kit combines multiple steps and omits multiple purification steps, thus significantly reducing the minimum amount of starting template DNA required and shortening the library construction time. In addition, the kit employs high-fidelity DNA polymerase for library enrichment and preference-free PCR amplification, which expands the region of sequence coverage and enables efficient preparation of DNA libraries for use in the Ion torrent second-generation sequencing platform.
Provide your own instruments, reagents and consumables
1．MAGNETIC FRAME
2．DNA Purification and Recovery Kit
3．Sample splice primer kit.
4．Anhydrous ethanol, EB (10 mM Tris-HCl, pH 8.0), deionized water (pH between 7.0 and 8.0).
5．Reaction tubes: It is recommended to use PCR tubes with low adsorption and 1.5 ml centrifuge tubes; Tips: It is recommended to use high-quality filtration tips to prevent contamination of kits and library samples.
Pre-experiment Preparation and Important Notes
1．Avoid repeated freezing and thawing of the Buffer in the kit, and it is recommended to store the Buffer in portions for the first use. The enzyme should be put back to -20℃ for storage as soon as possible after use.
2．PCR products due to improper operation is very easy to produce pollution, resulting in inaccurate experimental results, it is recommended that the PCR reaction system preparation PCR product purification area isolation, and the use of special pipettes, regular cleaning of the various experimental areas.
DNA end repair reaction:
1．Add the following components to a 200 μl PCR tube, gently mix the above solution with a lance tip, and centrifuge instantaneously to allow all components to collect at the bottom of the tube.
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2．The tubes were placed in the PCR instrument with the thermal cap open and the reaction program was as follows:
20min@25℃
10min@70℃
Hold on 4℃
Adaptor connection:
The following is the procedure for connecting with adapter:
1．Add the following reagents directly to the above reaction solution, mix the above reagents with a lance tip and centrifuge briefly so that the solution collects at the bottom of the tube.
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Note: It is recommended that the molar ratio of the amount of Adaptor added to the DNA fragments is 10:1-20:1, please refer to the following table for the specific concentration of Adaptor to be used. If the amount of DNA is 10-100ng, the recommended concentration of Adaptor is 1μM (less than 260bp) or 0.5μM (300-400bp).
2．reaction step
15min@25℃
5min@65℃
Hold on 4℃
Selective recovery of Adaptor ligated DNA fragments
Selective recovery of DNA fragments is required for the construction of DNA libraries of different sizes. If the starting sample size is lower than 50ng, selective recovery of DNA fragments is not recommended. Another solution can be referred to for direct purification of DNA fragments. The following procedure uses the Magnetic Bead Method DNA Purification and Recovery Kit, which can selectively recover DNA fragments in the length range of 310-370bp (read length of 200bp), and the starting volume of the reaction is 100μl.
1．Vortex-shake the CMPure for 20 seconds to thoroughly mix it into a homogeneous solution;
2．Transfer 100 μl of adaptor ligation reaction buffer to a new 1.5 ml centrifuge tube;
3．Add 60µl of well-mixed CMPure, vortex and shake for 5 seconds and let stand at room temperature for 5 minutes;
4．Centrifuge briefly, place the tube on a magnetic rack to separate the magnetic beads from the supernatant solution until the solution is clear (takes about 5 minutes), carefully transfer the supernatant solution to a new 1.5 ml centrifuge tube, and discard the magnetic beads;
Note: Do not discard the top clear.
5．Add 20µl of well-mixed CMPure to the supernatant, vortex and shake for 5 seconds and leave at room temperature for 5 minutes;
6．Centrifuge briefly, place the tube on a magnetic rack to separate the magnetic beads from the supernatant solution until the solution is clear (takes about 5 minutes), carefully remove the supernatant and discard it, avoiding contact with the magnetic beads that bind the target DNA during this time;
Note: Do not discard the beads.
7．Continuing to keep the centrifuge tube fixed on a magnetic rack, add 250µl of freshly configured 80% ethanol to the centrifuge tube and leave it at room temperature for 30 seconds and carefully discard the supernatant once the suspended magnetic beads are fully adsorbed;
8．Repeat step 7; for complete removal of residual liquid, the tube may be centrifuged briefly and the residual liquid removed again.
9．Keep the centrifuge tube fixed on a magnetic rack and let it stand at room temperature for 5 minutes to allow the magnetic beads to dry in the air;
10．Remove the centrifuge tube from the magnetic rack, add 25µl of 10mM Tris-HCl (pH 8.0) or deionized water (self-provided), vortex and oscillate to completely resuspend the magnetic beads in the eluate, and allow to stand at room temperature for 5 minutes;
11．Centrifuge briefly, place the tube on a magnetic rack until the solution is clear (takes about 5 minutes), and transfer 25 μl of the eluate to a new PCR tube;
Another option: full recovery of Adaptor ligated DNA fragments
1．Vortex-shake the CMPure for 20 seconds to thoroughly mix it into a homogeneous solution.
2．Transfer the adaptor ligation reaction solution to a new 1.5 ml centrifuge tube.
3．Add 1x the sample volume of CMPure, vortex and shake for 5 seconds and let stand at room temperature for 5 minutes.
4．Centrifuge briefly, place the tube on a magnetic rack to separate the magnetic beads from the supernatant solution until the solution is clear (takes about 5 minutes), carefully aspirate the supernatant and discard, avoiding contact with magnetic beads that have bound the target DNA during this time.
Note: Do not discard the beads.
5．Continuing to keep the centrifuge tube fixed on a magnetic rack, add 250 μl of freshly configured 80% ethanol to the centrifuge tube and leave it at room temperature for 30 s. After the suspended magnetic beads are fully adsorbed, carefully discard the supernatant.
6．Repeat step 5. To completely remove the residual liquid, centrifuge the tube briefly and then remove the residual liquid again.
7．Keep the centrifuge tube fixed on a magnetic rack and let it stand at room temperature for 5 minutes to allow the magnetic beads to dry in the air.
8．Remove the centrifuge tube from the magnetic rack, add 25 μl of EB (self-provided) or deionized water, vortex and oscillate to completely resuspend the magnetic beads in the eluent, and allow to stand for 5 minutes at room temperature.
9．Centrifuge briefly, place the tube on a magnetic rack until the solution is clear (takes about 5 minutes), and transfer 25 μl of the eluate to a new PCR tube.
PCR enrichment
1. Add the following reagents to the PCR tube and mix well
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2. PCR reaction conditions
Note: 4-6 cycles for a sample size of 1ug, 6-8 cycles for a sample size of 100ng, and 10-12 cycles for a sample size of 10ng.The number of PCR cycles can be optimized according to the experiment.
Purification of PCR products
1．Vortex-shake the CMPure for 20 seconds to thoroughly mix it into a homogeneous solution;
2．The PCR reaction solution was transferred to a new 1.5 ml centrifuge tube;
3．Add 1x the sample volume of CMPure, vortex and shake for 5 seconds and let stand at room temperature for 5 minutes;
4．Centrifuge briefly and place the tube on a magnetic rack to separate the magnetic beads from the supernatant solution until the solution is clear (takes about 5 minutes). Carefully remove and discard the supernatant, avoiding contact with the magnetic beads that bind the target DNA during this time; note: do not discard the magnetic beads!
5．Continuing to keep the centrifuge tube fixed on a magnetic rack, add 250µl of freshly configured 80% ethanol to the centrifuge tube and leave it at room temperature for 30 seconds and carefully discard the supernatant once the suspended magnetic beads are fully adsorbed.
6．Repeat step 5; for complete removal of residual liquid, the tube may be centrifuged briefly and the residual liquid removed again.
7．Keep the centrifuge tube fixed on a magnetic rack and let it stand at room temperature for 5 minutes to allow the magnetic beads to dry in the air.
8．Remove the centrifuge tube from the magnetic rack, add 25µl of EB (self-provided) or deionized water, vortex and oscillate to completely resuspend the magnetic beads in the eluent solution, let it stand at room temperature for 5 minutes, centrifuge briefly, place the tube on the magnetic rack until the solution is clear (takes about 5 minutes), transfer 25µl of elution solution into a new PCR tube, and store the DNA libraries at -20℃.
规格
别名
NGS 快速 DNA 文库制备套件
规格或纯度
for Ion Torrent
英文名称
NGS Fast DNA Library Prep Set
储存条件
-20°C储存,避免反复冻融
运输条件
超低温运输
产品线
生物产品
名称和识别符
分子类型
小分子
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
产品仅提供给经过验证的企业、机构和合格的专业人员用于研发。不适用于人类、动物、诊断或治疗。
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