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
2-8°C储存,-20°C储存。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
产品内容
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产品介绍
环化试剂盒是针对华大智造(MGI) 高通量测序平台量身打造的模块化试剂盒。使用本试剂盒可以将接头连接后的PCR产物制备成适用于MGI测序仪专用的单链环状DNA文库。试剂盒中提供的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。
自备仪器、试剂和耗材
1. 磁力架:建议使用DynaMagTM-2.
2.Qubit"3.0 荧光定量仪
3.Qubit" ssDNA Assay Kit
4.无水乙醇，EB(10 mM Tris-HCl, pH 8.0),NF Water(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 mIEP管:5.
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.样本准备:
·PCR 产物:总量2330 ng(多个 PCR产物混合时,是指混合后的总量)，体积49 pL(如果 PCR 产物本身体积不足,补充 NF Water 至总体积 49 pl)。·PCR 产物片段大小:片段主峰在 200-500 bp 之间。·PCR 产物修饰: 已加入适用于MGISEQ-2000、MGISEQ-200以及BGISEQ-500等测序平台的固定序列(含Index)
2.试剂准备
·取出试剂盒内对应试剂，短暂离心，酶混合液置于冰上待用:缓冲液使用前需在室温溶解后，震荡离心，置于冰上待用，NF Water和EB置于室温待用:”请在冰上配制混合液:
试剂盒内缓冲液溶解后可能出现沉淀，沉淀不影响试剂功能，请充分震荡混匀直至沉淀消失后使用。
环化流程示意图
操作步骤
环化
1. 向 49JI PCR 产物加入 1 wl Splint Oligo. 在 PCR仪上进行 95°C变性孵育3分钟，然后立即转移到冰浴，静置2min。
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2. 在冰上按如下体系配制反应混合液:
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3.向 50μl 变性后的 DNA 中加入上述 15ul 反应混合液:
4.将上述 PCR 管置于 PCR 仪上，如下条件反应:
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酶切消化
1. 在冰上按如下体系配制消化反应液:
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2.环化反应结束后，向环化体系中直接加入8l 消化反应液，混匀，短暂离心后将PCR 管置于 PCR 仪上，如下条件反应:
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3.反应结束后，立即进行纯化。
消化产物纯化
1.前 30 分钟取出 CMPure 置于室温，使用前充分震荡混匀:
2. 消化产物转移至1.5 mIEP管中，吸取340pICMPure 至消化产物中，使用移液器轻轻吹打10 次充分混匀，室温孵育10 分钟:
3.瞬时离心，将EP管置于磁力架，静置5分钟至液体澄清，移液器吸取并弃掉上清:
4.保持EP管固定于磁力架上，加入 250ul新鲜配制的80%乙醇,室温静置 1分钟后小心弃去上清:
5.重复步骤4 一次，尽量吸干管底液体:注意:不要吸取磁珠,以免影响产量,
6.保持EP管固定于磁力架上，打开管盖，室温干燥 5-10 分钟;
7.将EP管从磁力架上取下，加入35ul EB或NF Water进行 DNA 洗脱，移液器吹打混匀并室温下溶解10分钟:
8.瞬时离心，将EP管置于磁力架上，静置2分钟至液体澄清，将上清液转移到新EP管中。-20C保存，待制备DNB。
Products content
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Products Introduction
The Cyclization Kit is a modular kit tailored for the MGI high-throughput sequencing platform. With this kit, PCR products after junction ligation can be prepared into single-stranded circular DNA libraries suitable for MGI sequencers. All reagents provided in the kit have been subjected to stringent quality control and functional validation to maximize the stability and reproducibility of library construction.
Provide your own instruments, reagents and consumables
1. Magnetic frame: DynaMagTM-2 (Cat. No. 12321D) is recommended.
2. "Qubit" 3.0 Fluorescence Quantimeter (ThermoFisher, Cat. No. Q33216)
3. Qubit" ssDNA Assay Kit (Invitrogen, Cat. No. Q10212)
4. Anhydrous ethanol, EB (10 mM Tris-HCl, pH 8.0), NF Water (pH between 7.0 and 8.0).
5. reaction tubes: low adsorption PCR tubes with 1.5 mIEP tubes are recommended: 5.
Tip: It is recommended to use a high quality filter tip to prevent contamination of kits and libraries.
Pre-experiment Preparation and Important Notes
1. Sample preparation.
PCR product: 2330 ng total (total amount when multiple PCR products are mixed) in a volume of 49 pL (if the volume of PCR product is insufficient, add NF Water to bring the total volume to 49 pl). -PCR product: Fragment size: The fragment peak is between 200-500 bp. -PCR product fragment size: Fragment peaks between 200-500 bp. -PCR product modification: Fixed sequences (with Index) for MGISEQ-2000, MGISEQ-200 and BGISEQ-500 sequencing platforms were added.
2. Reagent preparation
-Remove the corresponding reagents from the kit, centrifuge briefly, and place the enzyme mixture on ice until ready to use: buffers need to be dissolved at room temperature before use, then centrifuged with shaking and placed on ice until ready to use, and NF Water and EB are placed at room temperature until ready to use: "Please make up the mixture on ice:
Precipitation may appear after the buffer in the kit is dissolved, the precipitation does not affect the function of the reagent, please shake and mix well until the precipitation disappears and then use.
Schematic diagram of the cyclization process
procedure
cyclize
1. 1 wl of Splint Oligo was added to the 49JI PCR product. The product was denatured and incubated on a PCR instrument at 95°C for 3 min, then immediately transferred to an ice bath and allowed to stand for 2 min.
2. The reaction mixture was prepared on ice according to the following system.
3. Add 15ul of the above reaction mixture to 50μl of denatured DNA.
4. Place the above PCR tubes on the PCR instrument under the following conditions Reaction.
digest
1. Prepare the digestion reaction solution on ice according to the following system.
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2. After the cyclization reaction, add 8l of digestion reaction solution directly to the cyclization system, mix well, centrifuge briefly and then place the PCR tube on the PCR instrument and react under the following conditions.
3. Purification was carried out immediately after the reaction.
Purification of digestive products
1. Remove CMPure at room temperature 30 minutes prior to use and mix well with shaking.
2. Transfer the digested product to a 1.5 mIEP tube, pipette 340 pICMPure into the digested product, mix well by gently blowing 10 times with a pipette and incubate for 10 minutes at room temperature.
3. Instantaneous centrifugation, place the EP tube on a magnetic rack and let stand for 5 minutes until the liquid is clear, pipette and discard the supernatant.
4. Keep the EP tube fixed on a magnetic rack, add 250ul of freshly prepared 80% ethanol, let it stand at room temperature for 1 minute, then carefully discard the supernatant.
5. Repeat step 4 once, try to suck up the liquid at the bottom of the tube: Note: Do not suck up the magnetic beads, so as not to affect the yield.
6. Keep the EP tube fixed on the magnetic rack, open the cap and dry it at room temperature for 5-10 minutes.
7. Remove the EP tube from the magnetic rack, add 35ul of EB or NF Water for DNA elution, pipette blow to mix and dissolve at room temperature for 10 min.
8. Centrifuge instantaneously, place the EP tube on a magnetic rack and let stand for 2 minutes until the liquid is clarified, transfer the supernatant to a new EP tube. -Store at 20C and leave to prepare DNB.
规格
英文名称
Circularization Kit For MGI
储存与运输
储存条件
2-8°C储存,-20°C储存
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:
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。需冷藏保存以维持规定的有效期。
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。
如何获取批次专用COA？
每个订单都会随附分析证书（COA），您也可以从账户订单历史中下载。如需订购前的COA，请联系您的客户代表，或使用PDP上的"申请COA"按钮——我们将提供代表性批次的COA。
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