| 货号 (SKU) | 包装规格 | 是否现货 | 价格 | 数量 |
|---|---|---|---|---|
| S665716-24T |
24T |
期货  |
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| S665716-96T |
96T |
期货 |
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| 别名 | 单细胞WGA试剂盒(MDA) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| 英文名称 | Single Cell WGA Kit（MDA） | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 储存温度 | -20°C储存,避免反复冻融 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 运输条件 | 超低温冰袋运输 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 产品介绍 |
产品简介 单细胞全基因组扩增试剂盒基于MDA的等温扩增体系，可以以单个细胞或者微量 样本为模板实现全基因组扩增。单细胞全基因组经扩增后扩增产物大小在2-100 kb间， 可广泛适用于二代测序、大片段拷贝数变异分析、微卫星分析、qPCR分析、基因芯片 分析等。 本试剂盒使用的Phi29 DNA聚合酶是从噬菌体中克隆的DNA聚合酶，具有很强的链 置换活性和链亲合力，单次聚合反应可以实现长达100 kb的连续聚合延伸，其扩增产物 适用于多种下游应用，Phi29 DNA聚合酶还具有很强的3’-5’外切酶活性，保证了DNA合 成的高保真性。正常情况下一个反应可以产生大于20μg高覆盖度的基因组DNA。 自备仪器、试剂 离心机 水浴锅或PCR仪 反应管：建议使用低吸附的PCR管 枪头：建议使用高质量过滤枪头防止污染 去离子水 注意事项 1．本产品检测灵敏度极高，实验操作应在正压的超净工作台中完成，扩增反应产物浓度 较高，应做好隔离，避免扩增产物导致的气溶胶污染。 2．以低质量的样品为模板会影响最终的扩增产物质量，应尽量避免使用大量降解和片段 化的DNA作为起始样本。 操作流程示意图 操作步骤 细胞为模板扩增 本方案适用于以1-1000个细胞为模板进行全基因组无差别扩增。应使用新鲜制备的细 胞样品，以保证起始基因组的完整性，请勿使用已发生凋亡的细胞。 1．准备Buffer D2(下表给出的Buffer D2体积足够12个反应，一次实验未完全用完可储存于-20°C ，但储存时间不能超过3个月)。
2．将4 μl细胞样品(重悬于PBS中)加入到PCR管中。 如果样品体积少于4 μl，请使用PBS 补足到4 μl。 3．加入3μl Buffer D2，轻弹管壁混匀并短暂离心收集。请确保细胞没有粘附在管壁上， 请勿使用移液器吹打，避免细胞样品粘附到移液器的吸头上。 4．样品65℃孵育10 min。 5．加入3 μl Buffer N，轻弹管壁混匀并短暂离心。在下步反应准备好之前请将样品置于 冰上。 6．按照下表准备反应混合液，混匀并短暂离心。
7．立即将40 μl反应混合液加入到准备好的10 μl DNA样品中(第5步)，轻弹管壁混匀并短 暂离心收集。 8．30℃孵育2h，如有需要可延长孵育时间增加产量。 9．65℃孵育5 min失活SC-DNA Polymerase。 注：扩增产物是高浓度基因组DNA，请使用水或者TE稀释到合适的浓度后进行下游实 验。扩增产物可广泛应用于全基因组和外显子测序、qPCR分析、基因芯片分析等。 基因组为模板扩增 本方案适用于大于1 ng纯化的基因组DNA为模板进行全基因组的无差别扩增，如果基因 组完整度与纯度足够高，更少的起始DNA也可以使用。 1．准备Buffer D1及N1(下表给出的体积足够12个反应，一次实验未完全用完可储存于 -20°C ，但储存时间不能超过3个月)。
2．将2.5 μl DNA样品加入到PCR管中，如果样品体积少于2.5 μl，请使用水或者TE补足 到2.5 μl。 3．加入2.5 μl Buffer D1，轻弹管壁混匀并短暂离心。 4．室温（15-25℃）孵育3 min。 5．加入5 μl Buffer N1，轻弹管壁混匀并短暂离心。下步反应准备好之前请将样品置于 冰上。 6．按照下表准备反应混合液，混匀并短暂离心。
7．立即将40 μl反应混合液加入到准备好的10 μl DNA样品中(第5步)，轻弹管壁混匀并短 暂离心收集。 8．30℃孵育2h，如有需要可延长孵育时间增加产量。 9．65℃孵育5 min失活SC-DNA Polymerase。
注：扩增产物是高浓度基因组DNA，请使用水或者TE稀释到合适的浓度后进行下游实
验。扩增产物可广泛应用于全基因组和外显子测序、qPCR分析、基因芯片分析等。
Products content
Products Introduction The Single Cell Whole Genome Amplification Kit is an isothermal amplification system based on MDA, which can be used as a template for whole genome amplification of single cells or micro samples. The size of single-cell whole genome amplification products ranges from 2-100 kb, which can be widely used in second-generation sequencing, large-band copy number variation analysis, microsatellite analysis, qPCR analysis, gene chip analysis and so on. The Phi29 DNA polymerase used in this kit is a DNA polymerase cloned from phage, which has strong strand displacement activity and strand affinity, and can achieve continuous polymerization and extension of up to 100 kb in a single polymerization reaction, and its amplification products are suitable for a variety of downstream applications, and the Phi29 DNA polymerase has strong 3'-5' exonuclease activity, and can be widely used in second-generation sequencing, copy number variation analysis of large fragments, microsatellite analysis, qPCR analysis and gene chip analysis. Phi29 DNA polymerase also has strong 3'-5' exonuclease activity, which ensures high fidelity of DNA synthesis. Under normal conditions, a single reaction can produce more than 20 μg of genomic DNA with high coverage. Bring your own instruments and reagents centrifuges Water bath or PCR instrument Reaction Tubes: Low adsorption PCR tubes are recommended. Gun Heads: High quality filtered gun heads are recommended to prevent contamination. deionized water
caveat 1. The sensitivity of this product is very high, the experimental operation should be completed in the positive pressure of the ultra-clean bench, the concentration of the amplification reaction product is high, should be isolated to avoid aerosol contamination caused by the amplification product. 2. The use of low-quality samples as templates can affect the quality of the final amplification product, and the use of heavily degraded and fragmented DNA as starting samples should be avoided. Operation flow diagram
procedure Cells as templates for amplification This protocol is suitable for whole genome non-discriminatory amplification using 1-1000 cells as a template. Freshly prepared cell samples should be used to ensure the integrity of the starting genome, and apoptotic cells should not be used. 1. This protocol is suitable for whole genome non-discriminatory amplification using 1-1000 cells as a template. Freshly prepared cell samples should be used to ensure the integrity of the starting genome, and apoptotic cells should not be used.
2. Add 4 μl of cell sample (resuspended in PBS) to the PCR tube. If the sample volume is less than 4 μl, make up to 4 μl using PBS. 3. Add 3 μl of Buffer D2, flick the wall of the tube to mix and centrifuge briefly to collect. Make sure that the cells are not adhering to the wall of the tube, and do not blow with the pipette to avoid the cell sample adhering to the pipette tip. 4. The samples were incubated at 65°C for 10 min. 5. Add 3 μl of Buffer N, flick the walls of the tube to mix and centrifuge briefly. Keep the sample on ice until ready for the next step. 6. Prepare the reaction mixture according to the table below, mix and centrifuge briefly.
7. Immediately add 40 μl of the reaction mixture to the prepared 10 μl DNA sample (step 5), flick the walls of the tube to mix and collect by brief centrifugation. 8. Incubate at 30°C for 2 h. Incubation can be extended to increase yield if needed. 9. Incubate at 65℃ for 5 min to inactivate SC-DNA Polymerase. Note: The amplified product is a high concentration of genomic DNA, please use water or TE to dilute to a suitable concentration for downstream experiments. The amplified product can be widely used in whole genome and exon sequencing, qPCR analysis, gene chip analysis, etc.
Genome as template amplification This protocol is suitable for non-discriminatory genome-wide amplification of greater than 1 ng of purified genomic DNA as a template, but less starting DNA can be used if the genome is sufficiently complete and pure. 1．Prepare Buffer D1 and N1 (the volumes given in the table below are sufficient for 12 reactions, and may be stored at -20°C if not fully used in one experiment, but should not be stored for more than 3 months).
2．Add 2.5 μl of DNA sample to the PCR tube. If the sample volume is less than 2.5 μl, use water or TE to make up to 2.5 μl. 3．Add 2.5 μl of Buffer D1, flick the tube wall to mix and centrifuge briefly. 4．Incubate at room temperature (15-25°C) for 3 min. 5．Add 5 μl of Buffer N1, flick the walls of the tube to mix and centrifuge briefly. Keep the sample on ice until ready for the next step. 6．Prepare the reaction mixture according to the table below, mix and centrifuge briefly.
7．Immediately add 40 μl of the reaction mixture to the prepared 10 μl DNA sample (step 5), flick the walls of the tube to mix and collect by brief centrifugation. 8．Incubate at 30°C for 2 h. Incubation can be extended to increase yield if needed. 9．SC-DNA Polymerase was inactivated by incubation at 65°C for 5 min. Note: The amplified product is a highly concentrated genomic DNA, please use water or TE to dilute it to a suitable concentration for downstream experiments. The amplified product can be widely used in whole genome and exon sequencing, qPCR analysis, gene chip analysis and so on.
Usage The following are examples of conventional PCR reaction systems and conditions, which should be improved and optimized according to the template, primer structure and fragment size.
1. PCR Reaction System All operations should be carried out on ice, and the components should be mixed well after thawing and stored at -20℃ after use.
2. PCR reaction conditions
take note of 1）Priority is given to three-step amplification; if the three-step method fails to amplify the target product or if the primer Tm value is greater than 68°C, try the two-step method. 2）Denaturation: pre-denaturation of simple templates 98°C, 30s-1min, for complex templates, the pre-denaturation time can be extended to 3min. 3）Annealing: In general, the annealing temperature is 3-5℃ lower than the Tm value of the primers. If the desired amplification efficiency cannot be obtained, the annealing temperature should be changed in a gradient to optimize the results; if a non-specific reaction occurs, the annealing temperature should be increased appropriately. 4）Extension: The extension time should be set according to the length of the amplified fragments and the complexity of the template, the amplification efficiency of this product is 3-5 kb/min, for long fragments and templates with high complexity it is recommended that 2-4kb/min. 5）Cycling times: the number of cycles can be set according to the downstream application of the amplification product. If the number of cycles is too small, the amplification amount will be insufficient, and if the number of cycles is too large, the chance of mismatch will be increased, so the number of cycles should be minimized under the premise of guaranteeing the yield of the product.
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