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| 别名 | HiFiScript cDNA 合成试剂盒 | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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| 英文名称 | HiFiScript cDNA Synthesis Kit | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| 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 本产品是专为两步法RT-PCR第一步实验配制的cDNA第一链合成试剂盒。该试剂盒中使用的HiFiScript逆转录酶是M-MLV由来的新型高效逆转录酶，新颖突变位点大幅提升酶的转录活性，cDNA第一链合成的效率和产量更高，可利用pg级的总RNA或mRNA合成cDNA第一链。点突变消除RNase H活性，酶的延伸性能提高，有效改善逆转录酶与RNA模板的亲和力，可获得长至12 kb的cDNA。精心优化的RT Buffer使逆转录酶应用范围更广，对后续的PCR以及定量PCR实验兼容性高。该试剂盒配备所有逆转录试剂，使用简单方便。适用于第一链cDNA的合成和后续的RT-PCR、RT-qPCR，以及全长cDNA文库的构建等。 产品特点 1．高效的逆转录效率：新颖突变位点大幅提升酶活性能，获得更高产量的cDNA。 2．良好的延伸能力：点突变消除RNase H活性，改善逆转录酶与RNA模板的亲和力，可获得长至12 kb的cDNA。 3．灵敏度高：可利用pg级的总RNA或mRNA模板合成cDNA第一链。 4．使用方便：逆转录试剂盒中已配备所有逆转录试剂，仅需准备模板即可轻松完成逆转录。 注意事项 1．在操作过程中应避免RNase污染，防止RNA降解或实验中的交叉污染，建议在专门的区域进行RNA操作，使用专门的仪器和耗材，操作人员带口罩和一次性手套并经常更换手套。 2．实验尽量使用一次性塑料器皿，若使用玻璃器皿，应使用0.1％DEPC（焦碳酸二乙酯）水溶液在37℃处理12小时，并在120℃下高压灭菌30分钟后使用，或者将玻璃器皿在180℃下干热灭菌60分钟后使用。实验中用到的无菌水应使用0.1%的DEPC处理后进行高压灭菌。 3．本试剂盒中的所有试剂使用前请上下颠倒轻轻混匀，尽量避免起泡，并经短暂离心后使用。所涉及的酶类使用后应尽快放回-20℃，避免反复冻融。 4．若起始RNA的量小于50 ng，建议加入RNA酶抑制剂（RNasin）。 使用方法 注意：1 ng-5 μg总RNA可建立20 μl反应体系，如果总RNA量大于5 μg，请按比例扩大反应体系。 i 逆转录操作步骤： 1．将RNA模板、Primer Mix、dNTP Mix、DTT、RT Buffer、HiFiScript和RNase-Free Water溶解并置于冰上备用。 2．根据以下表格配制反应体系，总体积为20 μl。
注意：1）若起始RNA的量小于50 ng，则建议加入RNA酶抑制剂（RNasin）。 2）Primer Mix由Oligo(dT)和Random Primer配制而成。 3．涡旋震荡混匀，短暂离心，使管壁上的溶液收集到管底。 4．cDNA合成反应条件： 1）若下游进行荧光定量PCR检测，42℃孵育15分钟，85℃孵育5分钟。 2）若下游进行普通PCR检测，42℃孵育30-50分钟，85℃孵育5分钟。 注意：对于二级结构复杂或GC含量高的模板，可以提高逆转录温度至50℃，增强逆转录效率。 5．反应结束后，短暂离心，置于冰上冷却。 6．逆转录产物可直接用于PCR反应和荧光定量PCR反应，或置于-20℃长期保存。 ii 若逆转录效率低，或RNA模板二级结构复杂、GC含量高时，建议采用以下步骤： 1．将RNA模板、Primer Mix、dNTP Mix、DTT、RT Buffer、HiFiScript和RNase-Free Water溶解并置于冰上备用。 2．根据以下表格配制反应体系，总体积为13 μl 。
3．70℃孵育10分钟，迅速冰浴2分钟。 4．短暂离心，使管壁上的溶液收集到管底。 5．继续向以上反应液中加入以下试剂：
注意：1）若起始RNA的量小于50 ng，则建议加入RNA酶抑制剂（RNasin）。 2）Primer Mix由Oligo（dT）和Random primer配制而成。 6．进行cDNA第一链合成： 1）若下游进行荧光定量PCR检测，50℃孵育15分钟，85℃孵育5分钟。 2）若下游进行普通PCR检测，50℃孵育30-50分钟，85℃孵育5分钟。 7．反应结束后，短暂离心，置于冰上冷却。 8．逆转录产物可直接用于PCR反应和荧光定量PCR反应，加入量应小于PCR反应体系的1/10，或置于-20℃长期保存。
Product Introduction This product is a cDNA first-strand synthesis kit specially formulated for the first step of two-step RT-PCR. The HiFiScript Reverse Transcriptase used in this kit is a new high-efficiency reverse transcriptase derived from M-MLV, with novel mutation sites that dramatically improve the transcriptional activity of the enzyme, and the efficiency and yield of cDNA first-strand synthesis is higher, and the first strand of cDNA can be synthesized using pg-level total RNA or mRNA. The point mutation eliminates RNase H activity and improves the elongation performance of the enzyme, which effectively improves the affinity of the reverse transcriptase to the RNA template, and allows for the production of cDNAs up to 12 kb. The carefully optimized RT Buffer allows for a wider range of applications of the reverse transcriptase, and provides high compatibility for subsequent PCR and quantitative PCR experiments. The kit comes with all reverse transcription reagents and is easy to use. It is suitable for first-strand cDNA synthesis and subsequent RT-PCR, RT-qPCR, and full-length cDNA library construction. Product Features 1. Highly efficient reverse transcription efficiency: novel mutation sites dramatically increase enzyme activity for higher yields of cDNA. 2. Good elongation: point mutation eliminates RNase H activity and improves the affinity of reverse transcriptase for RNA templates, resulting in cDNAs up to 12 kb long. 3. High sensitivity: cDNA first strand can be synthesized using pg-level total RNA or mRNA templates. 4. Easy to use: The Reverse Transcription Kit comes with all the reverse transcription reagents, so you can easily complete the reverse transcription by simply preparing the template. matters needing attention 1. RNase contamination should be avoided during operation to prevent RNA degradation or cross-contamination in the experiment. It is recommended that RNA operations be performed in a dedicated area, specialized instruments and consumables be used, and that operators wear masks and disposable gloves and change gloves frequently. 2. Disposable plasticware should be used as much as possible. If glassware is used, it should be treated with 0.1% DEPC (diethylpyrocarbonate) aqueous solution at 37℃ for 12 hours and autoclaved at 120℃ for 30 minutes before use, or the glassware should be dry heat sterilized at 180℃ for 60 minutes before use. Sterile water used in the experiment should be treated with 0.1% DEPC and then autoclaved. 3. All reagents in this kit should be mixed gently before use by turning up and down to avoid foaming as much as possible, and after brief centrifugation. The enzymes involved should be put back to -20℃ as soon as possible after use to avoid repeated freezing and thawing. If the amount of starting RNA is less than 50ng, it is recommended to add RNAase inhibitor (RNasin). Usage Note: 1ng-5μg of total RNA can establish a 20μl reaction system, if the amount of total RNA is greater than 5μg, please expand the reaction system proportionally. Reverse transcription procedure: 1. Dissolve RNA template, Primer Mix, dNTP Mix, DTT, RT Buffer, HiFiScript and RNase-Free Water and set aside on ice. 2. Prepare the reaction system according to the following table in a total volume of 20 μl.
Note: 1) If the amount of starting RNA is less than 50ng, it is recommended to add RNAase inhibitor (RNasin). 2) Primer Mix was prepared from Oligo (dT) and Random Primer. 3. Vortex and shake to mix and centrifuge briefly so that the solution on the walls of the tube collects at the bottom. 4. cDNA synthesis reaction conditions: 1) If fluorescent quantitative PCR assay is performed downstream, incubate at 42°C for 15 minutes and 85°C for 5 minutes. 2) If downstream for normal PCR assay, incubate at 42°C for 30-50 minutes and 85°C for 5 minutes. Note: For templates with complex secondary structure or high GC content, the reverse transcription temperature can be increased to 50°C to enhance reverse transcription efficiency. 5. At the end of the reaction, centrifuge briefly and place on ice to cool. 6. The reverse transcription product can be directly used in PCR reaction and fluorescence quantitative PCR reaction, or placed in -20℃ for long-term storage. The following steps are recommended if reverse transcription efficiency is low, or if the RNA template secondary structure is complex and GC content is high: 1. Dissolve RNA template, Primer Mix, dNTP Mix, DTT, RT Buffer, HiFiScript and RNase-Free Water and set aside on ice. 2. Prepare the reaction system according to the following table in a total volume of 13 μl
3. Incubate at 70°C for 10 minutes and rapidly ice bath for 2 minutes. 4. Centrifuge briefly so that the solution on the walls of the tube collects at the bottom. 5. Continue to add the following reagents to the above reaction solution:
Note: 1) If the amount of starting RNA is less than 50ng, it is recommended to add RNAase inhibitor (RNasin). 2) Primer Mix was prepared from Oligo (dT) and Random primer. 6. Perform cDNA first-strand synthesis: 1) If fluorescent quantitative PCR assay is performed downstream, incubate at 50°C for 15 minutes and 85°C for 5 minutes. 2) If downstream for normal PCR assay, incubate at 50°C for 30-50 minutes and 85°C for 5 minutes. 7. At the end of the reaction, centrifuge briefly and place on ice to cool. 8. The reverse transcription product can be used directly in PCR reaction and fluorescence quantitative PCR reaction, the amount added should be less than 1/10 of the PCR reaction system, or placed in -20℃ for long-term storage. |
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