Table of Contents 目录 Introduction 1 Limitations of the Product 1 Materials Provided and Storage 3 Other Supplies Required 3 Precaution 5 QC Testing 5

Transcription

1 This package insert must be read entirely before using this product. For proper performance, use the insert provided with each individual product received. Catalog Number MR MR For fast quantitative real-time PCR with high sensitivity and high specificity. 适用于快速进行定量实时荧光 PCR, 灵敏度高 特异性高 2 SYBR Green Master Mix For research use only. Not for use in diagnostic procedures. 仅用于科研, 不得用于临床诊断 05/2017

2 Table of Contents 目录 Introduction 1 Limitations of the Product 1 Materials Provided and Storage 3 Other Supplies Required 3 Precaution 5 QC Testing 5 Protocol For ABI 7300, 7500, 7500 Fast and Step One Plus TM 7, 9, 11 For LightCycler and LightCycler , 13, 15 For Smart Cycler II System, CFX96 TM Real-time PCR Detection System and Thermal Cycler Dice Real Time System 17 PCR Optimization 19, 21 Troubleshooting 23, 25, 27 Appendix 29 Ordering Information 31 产品介绍 2 产品的局限 2 提供的材料及贮存 4 未提供的材料设备 4 注意事项 6 质控 6 实验流程 ABI Fast 和 Step One Plus TM 8, 10, 12 LightCycler 和 LightCycler , 14, 16 Smart Cycler II System CFX96 TM Real-time PCR Detection System 和 Thermal Cycler Dice Real Time System 18 PCR 优化 20, 22 常见问题与解决方案 24, 26, 28 附录 30 订购信息 32

3 INTRODUCTION 2 SYBR Green Master Mix is a 2 pre-mixed concentrate designed for real-time PCR using SYBR Green I chimeric fluorescence method, providing accurate real-time quantification of DNA and cdna targets in an easy-to-handle format. It contains HotStart Taq DNA polymerase, dntp mix, Mg 2+, SYBR Green I and PCR enhancer. High specificity and sensitivity in PCR are achieved by the use of the hot-start enzyme HotStar Taq DNA Polymerase together with a specialized PCR buffer. The product would produce good standard curves in a broad range and quantitative detect target genes accurately with good reproducibility and high reliability. SYBR Green I binds all double-stranded DNA molecules, emitting a fluorescent signal on binding, enabling the analysis of many different targets without having to synthesize target-specific labeled probes. The excitation and emission maxima of SYBR Green I are at 494 nm and 521 nm, respectively, which are compatible with use on any real-time cycler. HotStart Taq DNA Polymerase is provided in an inactive state and has no enzymatic activity at ambient temperature. This prevents the formation of misprimed products and primer-dimers during reaction setup and the first denaturation step, leading to high PCR specificity and accurate quantification. For certain thermal cyclers, ROX reference dye is required to compensate for non-pcr-related variations in fluorescence detection. 产品介绍 2 SYBR Green Master Mix 是 2 倍预混的浓缩液, 使用 SYBR Green I 嵌合荧光法进行 real-time PCR, 可简捷精确地对 DNA 和 cdna 靶序列进行实时荧光测定 本产品含有热启动 Taq DNA 聚合酶 dntp 混合物 Mg 2+ SYBR Green I 和 PCR 增强剂 热启动 Taq DNA 聚合酶结合独特的 PCR 缓冲液, 可实现高特异性 高灵敏度的 PCR 检测 本产品可在宽范围内得到良好的标准曲线, 对靶基因进行准确定量检测, 重复性好 可信度高 SYBR Green I 与所有双链 DNA 分子结合, 一结合即产生荧光信号, 可用于检测不同靶序列的检测, 而不需要合成靶序列特异性标记的探针 SYBR Green I 的最大激发波长和最大发射波长分别为 494 nm 和 521 nm, 可与任何实时荧光定量 PCR 仪兼容 热启动 Taq DNA 聚合酶为未活化状态, 在环境温度下不会展现酶活性 这可在反应加样和第一次变性阶段抑制错误引发和引物二聚体的形成, 大大提高 PCR 的高度特异性和准确定量 对于某些热循环仪, 需要加入 ROX 参考染料来校准荧光检测中的非 PCR 相关的差异 LIMITATIONS OF THE PRODUCT The product is intended for molecular biology applications. FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC PROCEDURES. The product should not be used beyond the expiration date on the label. Do not mix reagents with those from other lots or sources. 产品的局限 1. 本产品用于分子生物学实验, 仅用于科学研究, 非诊断试剂, 不能用于临床诊断 2. 请在本产品标记的有效期内使用 3. 本产品的试剂不能与其他批号的试剂或其他来源的试剂混合使用 1 2

4 MATERIALS PROVIDED AND STORAGE 提供的材料和贮存 Components Product Code SYBR Green Master Mix MR ml 1 ml 5 50 ROX Reference Dye MR μl 200 μl ddh 2O MR ml 1 ml 5 组分 编号 SYBR Green Master Mix MR ml 1 ml 5 50 ROX Reference Dye MR μl 200 μl 蒸馏水 MR ml 1 ml 5 Note: All reagents can be stored protected from light at 2-8 for 6 months or at -80 for long-term storage. Avoid repeat freezing-thaw. ROX Reference Dye is used to compensate for differences in fluorescence detection between wells due to slight variations in reaction volume or to differences in well position. Add 1/50 50 ROX Reference Dye for ABI 5700, 7000, 7300, 7700, 7900, 7900HT, 7900HT Fast, StepOne, StepOne Plus, etc. Add 1/ ROX Reference Dye for ABI 7500, 7500 Fast, ViiA 7, Stratagene MX4000, MX3005P, MX3000P, etc. No ROX required for Bio-Rad CFX96, CFX384, icycler iq, iq 5, MyiQ, MiniOpticon, Opticon, Opticon 2, Chromo4, Cepheid SmartCycler, Eppendorf Mastercycler ep realplex, realplex 2, Illumina Eco qpcr, Qiagen/Corbett Rotor-Gene Q, Rotor-Gene 3000, Rotor-Gene 6000, Roche Applied Science LightCycler TM 480, Thermo Scientific PikoReal Cycler, etc. 注 : 所有试剂可在 2-8 避光保存 6 个月, 或 -80 长期保存 避免反复冻融 由于反应体积的轻微差异或孔的位置差异,ROX Reference Dye 可用于校准孔与孔之间的荧光检测差异 以下仪器加 1/50 的 50 ROX Reference Dye:ABI HT 7900HT Fast StepOne StepOne Plus 等 以下仪器加 1/250 的 50 ROX Reference Dye:ABI Fast ViiA 7 Stratagene MX4000 MX3005P MX3000P 等 以下仪器无需 ROX Reference Dye:Bio-Rad CFX96 CFX384 icycler iq iq 5 MyiQ MiniOpticon Opticon Opticon 2 Chromo4 Cepheid SmartCycler Eppendorf Mastercycler ep realplex realplex 2 Illumina Eco qpcr Qiagen/Corbett Rotor-Gene Q Rotor-Gene 3000 Rotor-Gene 6000 Roche Applied Science LightCycler TM 480 Thermo Scientific PikoReal Cycler 等 OTHER SUPPLIES REQUIRED 1. Ice 2. Primers (Lyophilized primers should be dissolved in TE to provide a stock solution of 100 μm. Primer stock solutions should be stored in aliquots at -20 ) 3. Tubes or plates for real-time PCR 4. Pipet tips (pipet tips with aerosol barriers for preventing cross-contamination are recommended) 5. Real-time PCR thermal cycler 6. Centrifuge (with rotor for PCR tubes or plate) 未提供的材料设备 1. 冰 2. 引物 ( 引物粉末溶于 TE, 制备成 100 μm 储存液, 分装冻存于 -20 ) 3. Real-time PCR 反应管或反应板 4. 吸头 ( 推荐使用可防止气溶胶污染的吸头 ) 5. Real-time PCR 热循环仪 6. 离心机 ( 配 PCR 管或 PCR 板的转子 ). 3 4

5 PRECAUTION 1. Reagents are intended for research use only and are not for use in diagnostic or therapeutic procedures. 2. Do not mix or substitute reagents with those from other lots or other sources. 3. Do not use reagents beyond expiration date on label. 4. We therefore recommend that this product is handled only by those persons who have been trained in laboratory techniques and that it is used in accordance with the principles of good laboratory practice. Wear suitable protective clothing such as laboratory overalls, safety glasses, mask and gloves. 5. Do not eat or smoke in areas where reagents or samples are handled. 6. Use disposable tips containing hydrophobic filters to minimize cross-contamination 7. The product could not be used with TaqMan probes for quantitative real-time PCR. 8. The product contains SYBR Green I and ROX dye, avoid exposure to light during storage and preparation of PCR reactions. 9. When preparing PCR reaction, place the reagents on ice. 10. When stored at -80, white or light yellow precipitates may appear in 2 SYBR Green Master Mix, which make solution uniform. Dissolve precipitates slowly by palm temperature. Mix gently and thoroughly by inverting the tube up and down, do not vortex, and avoid bubbling, brief centrifuge before use. 注意事项 1. 本产品用于科学研究, 不能用于诊断治疗 2. 请不要使用其他批号或其他来源的试剂替代本产品中的试剂 3. 请不要使用过期的试剂 4. 联科生物推荐只有经过良好实验室培训的工作人员方可操作本产品 操作时请穿戴合适的防护设施, 例如实验服 安全眼镜 口罩和手套等 5. 在操作试剂或处理样本的区域请不要饮食 6. 使用具有疏水过滤的一次性吸头, 减少交叉污染 7. 本产品不能用于 TaqMan 探针法实时荧光定量 PCR 8. 本产品含有 SYBR Green I 和 ROX 染料, 保存和制备 PCR 反应液时注意避免强光照射 9. 制备 PCR 反应液时, 将试剂置于冰上 SYBR Green Master Mix 保存于 -80 时, 可能会产生白色或淡黄色沉淀, 导致溶液不均一 可用手温溶解沉淀 温和地上下颠倒管子充分混匀, 切勿涡旋, 避免起泡, 使用前简单离心 QC Testing Each lot of MultiSciences product is carefully tested in the quality control team. 质控 联科生物每个批次的产品都由质控部门进行严格的检测 5 6

6 PROTOCOL Important points to know For the highest efficiency in real-time PCR using SYBR Green I, targets should ideally be bp in length. The PCR must start with an initial incubation step of 30 seconds at 95 to activate HotStart Taq DNA Polymerase. Always readjust the threshold value for analysis of every run. 实验流程实验须知为了最高效地使用 SYBR Green I 进行 real-time PCR 检测, 理想的靶序列长度应在 bp 之间 PCR 的初始阶段应设置 秒的程序以活化热启动 Taq DNA 聚合酶 每次程序运行后进行分析时, 都需要重新调整阈值 Procedure For ABI 7300, 7500, 7500 Fast and Step One Plus TM thermal cyclers 1. Set up real-time PCR reactions on ice according to the following table. Note: Prepare a volume of mixture 10 % greater than that required for the total number of PCR assays to be performed. A negative control (without template DNA) should always be included. Component Volume/Reaction Volume/Reaction 2 SYBR Green Master Mix 10.0 μl 25.0 μl PCR Forward Primer (10 μm)* 0.4 μl 1.0 μl PCR Reverse Primer (10 μm)* 0.4 μl 1.0 μl Template DNA (<100 ng) # 2.0 μl 4.0 μl 50 ROX Reference Dye 0.4 μl or 0.08 μl 1 μl or 0.2 μl ddh 2O 6.8 μl or 7.12 μl 18 μl or 18.8 μl Total Volume 20.0 μl 50.0 μl * A final primer concentration of 0.2 μm is usually optimal. However, for individual determination of optimal primer concentration, a primer titration from 0.1 μm to 1 μm can be performed. # Generally, template DNA should not exceed than 100 ng in a 20 μl reaction. Determine the optimal usage of template DNA by gradient dilution due to the different copies of targets in different template DNA. For two-step RT-PCR, the volume of the cdna added (from the undiluted RT reaction) should not exceed 10% of the final PCR volume. For ABI 7300 and Step One Plus, add 1/50 50 ROX Reference Dye per reaction. For ABI 7500 and 7500 Fast, add 1/ ROX Reference Dye per reaction. Volume per reaction should be chosen according to the instruction provided by real-time PCR thermal cycler. 操作步骤 对于 ABI Fast 和 Step One Plus TM 热循环仪 1. 根据下表在冰上配制 real-time PCR 反应液 注意 : 根据反应总数计算所需的理论体积, 并额外多制备 10 % 的体积 实验中应始终设立不 含 DNA 模板的阴性对照 组分 每个反应的体积 每个反应的体积 2 SYBR Green Master Mix 10.0 μl 25.0 μl PCR Forward Primer (10 μm)* 0.4 μl 1.0 μl PCR Reverse Primer (10 μm)* 0.4 μl 1.0 μl Template DNA (<100 ng) # 2.0 μl 4.0 μl 50 ROX Reference Dye 0.4 μl or 0.08 μl 1 μl or 0.2 μl ddh 2O 6.8 μl or 7.12 μl 18 μl or 18.8 μl 总体积 20.0 μl 50.0 μl * 通常引物的终浓度为 0.4 μm 是最佳的 但对于个别需要确定最佳引物浓度的实验, 可将引物从 0.1 μm 到 1 μm 进行梯度摸索 # 通常在 20 μl 反应体系中, 模板 DNA 不应超过 100 ng 由于不同模板 DNA 中的靶序列拷贝数不同, 可通过梯度稀释模板 DNA 来确定最佳用量 对于两步的 RT-PCR, 从逆转录反应中得到的未稀 释的 cdna 的体积不应超过最终 PCR 体积的 10 % 对于 ABI 7300 和 Step One Plus, 每个反应加入 1/50 的 50 ROX Reference Dye 对于 ABI 7500 和 7500 Fast, 每个反应加入 1/250 的 50 ROX Reference Dye 根据 real-time PCR 热循环仪来选择反应体系 7 8

7 2. Set up real-time PCR program. Set up two-step PCR program according to the following graph is recommended. Determine the optimal PCR condition if necessary. Perform three-step PCR program when it is difficult to amplify targets using primers with low T m. For ABI 7300, 7500 and Step One Plus: 2. 设置 PCR 程序 建议根据下表设置两步法 PCR 程序 如有必要, 可优化 PCR 条件 当使用 T m 值较低的 引物, 难以扩增靶序列时, 可使用三步法 PCR 程序 对于 ABI 7300, 7500 和 Step One Plus: 阶段 程序 保温阶段 95,30 秒 循环阶段 变性 95,5 秒 (40 个循环 ) 退火 / 延伸 60,30-34 秒 * 熔解曲线阶段 * 对于 Step One Plus, 设为 30 秒 ; 对于 ABI 7300, 设为 31 秒 ; 对于 ABI7500, 设为 34 秒 Stage Program 对于 ABI 7500 Fast: Holding stage 95 for 30 seconds Cycling Stage Denaturation 95 for 5 seconds (40 cycles) Annealing/ Extension 60 for seconds* Melt Curve Stage * For Step One Plus, set at 30 seconds; for ABI 7300, set at 31 seconds; for ABI 7500, set at 34 seconds. For ABI 7500 Fast: 阶段保温阶段循环阶段 (40 个循环 ) 熔解曲线阶段 变性 退火 / 延伸 程序 95,30 秒 95,3 秒 60,30 秒 Stage Holding stage Cycling Stage (40 cycles) Melt Curve Stage Denaturation Annealing/ Extension Program 95 for 30 seconds 95 for 3 seconds 60 for 30 seconds 9 10

8 3. Analysis Check amplification plot and melt curve after reaction finish, make standard curve when performing absolute quantification of gene expression. 3. 结果分析 反应结束后, 确认 real-time PCR 的扩增曲线和熔解曲线, 对基因表达进行绝对定量时, 需制作标准曲线 For LightCycler and LightCycler 480 thermal cyclers 1. Set up real-time PCR reactions on ice according to the following table. Note: Prepare a volume of mixture 10 % greater than that required for the total number of PCR assays to be performed. A negative control (without template DNA) should always be included. Component Volume/Reaction 2 SYBR Green Master Mix 10.0 μl PCR Forward Primer (10 μm)* 0.4 μl PCR Reverse Primer (10 μm)* 0.4 μl Template DNA (<100 ng) # 2.0 μl ddh 2O 7.2 μl Total Volume 20.0 μl * A final primer concentration of 0.2 μm is usually optimal. However, for individual determination of optimal primer concentration, a primer titration from 0.1 μm to 1 μm can be performed. # Generally, template DNA should not exceed than 100 ng in a 20 μl reaction. Determine the optimal usage of template DNA by gradient dilution due to the different copies of targets in different template DNA. For two-step RT-PCR, the volume of the cdna added (from the undiluted RT reaction) should not exceed 10% of the final PCR volume. 对于 LightCycler 和 LightCycler 480 热循环仪 1. 根据下表在冰上配制 real-time PCR 反应液 注意 : 根据反应总数计算所需的理论体积, 并额外多制备 10 % 的体积 实验中应始终设立不含 DNA 模板的阴性对照 组分每个反应的体积 2 SYBR Green Master Mix 10.0 μl PCR Forward Primer (10 μm)* 0.4 μl PCR Reverse Primer (10 μm)* 0.4 μl Template DNA (<100 ng) # 2.0 μl ddh 2O 7.2 μl 总体积 20.0 μl * 通常引物的终浓度为 0.4 μm 是最佳的 但对于个别需要确定最佳引物浓度的实验, 可将引物从 0.1 μm 到 1 μm 进行梯度摸索 # 通常在 20 μl 反应体系中, 模板 DNA 不应超过 100 ng 由于不同模板 DNA 中的靶序列拷贝数不同, 可通过梯度稀释模板 DNA 来确定最佳用量 对于两步的 RT-PCR, 从逆转录反应中得到的未稀释的 cdna 的体积不应超过最终 PCR 体积的 10 % 2. Set up real-time PCR program. Set up two-step PCR program according to the following graph is recommended. Determine the optimal PCR condition if necessary. Perform three-step PCR program when it is difficult to amplify targets using primers with low T m. 2. 设置 PCR 程序 建议根据下表设置两步法 PCR 程序 如有必要, 可优化 PCR 条件 当使用 T m 值较低的 引物, 难以扩增靶序列时, 可使用三步法 PCR 程序 11 12

9 For LightCycler : 对于 LightCycler 阶段变性 PCR (40 个循环 ) 熔解曲线 变性 退火 / 延伸 程序 95,30 秒,20 / 秒 95, 5 秒,20 / 秒 60,30 秒,20 / 秒 95,0 秒,20 / 秒 65,25 秒,20 / 秒 95,0 秒,0.1 / 秒 Stage Denature PCR Denaturation (40 cycles) Annealing/ Extension Melting Curve Program 95 for 30 seconds, 20 /second 95 for 5 seconds, 20 /second 60 for 30 seconds, 20 /second 95 for 0 seconds, 20 /second 65 for 25 seconds, 20 /second 95 for 0 seconds, 0.1 /second 13 14

10 For LightCycler 480: 对于 LightCycler 480: 阶段预孵育扩增 (40 cycles) 熔解曲线降温 变性 退火 / 延伸 程序 95,30 秒,4.4 / 秒 95,5 秒,4.4 / 秒 60,30 秒,2.2 / 秒 95,5 秒,4.4 / 秒 65,60 秒,2.2 / 秒 95,0.11 / 秒 50,30 秒,2.2 / 秒 Stage Pre-incubation Amplification (40 cycles) Melting Curve Cooling Denaturation Annealing/ Extension Program 95 for 30 seconds, 4.4 /second 95 for 5 seconds, 4.4 /second 60 for 30 seconds, 2.2 /second 95 for 5 seconds, 4.4 /second 65 for 60 seconds, 2.2 /second 95 for 0 seconds, 0.11 /second 50 for 30 seconds, 2.2 /second 3. 结果分析 反应结束后, 确认 real-time PCR 的扩增曲线和熔解曲线, 对基因表达进行绝对定量时, 需制 作标准曲线 3. Analysis Check amplification plot and melt curve after reaction finish, make standard curve when performing absolute quantification of gene expression

11 For Smart Cycler II System, CFX96 TM Real-time PCR Detection System and Thermal Cycler Dice Real Time System thermal cyclers 1. Set up real-time PCR reactions on ice according to the following table. Note: Prepare a volume of mixture 10 % greater than that required for the total number of PCR assays to be performed. A negative control (without template DNA) should always be included. Component Volume/Reaction 2 SYBR Green Master Mix 12.5 μl PCR Forward Primer (10 μm)* 0.5 μl PCR Reverse Primer (10 μm)* 0.5 μl Template DNA (<100 ng) # 2.0 μl ddh 2O 9.5 μl Total Volume 25.0 μl * A final primer concentration of 0.2 μm is usually optimal. However, for individual determination of optimal primer concentration, a primer titration from 0.1 μm to 1 μm can be performed. # Generally, template DNA should not exceed than 100 ng in a 20 μl reaction. Determine the optimal usage of template DNA by gradient dilution due to the different copies of targets in different template DNA. For two-step RT-PCR, the volume of the cdna added (from the undiluted RT reaction) should not exceed 10% of the final PCR volume. 2. Set up real-time PCR program. Set up two-step PCR program according to the following graph is recommended. Determine the optimal PCR condition if necessary. Perform three-step PCR program when it is difficult to amplify targets using primers with low T m. Stage Program Pre-denature 95 for 30 seconds PCR Denaturation 95 for 5 seconds (40 cycles) Annealing/ Extension 60 for seconds Melting Curve 3. Analysis Check amplification plot and melt curve after reaction finish, make standard curve when performing absolute quantification of gene expression. 对于 Smart Cycler II System, CFX96 TM Real-time PCR Detection System and Thermal Cycler Dice Real Time System 热循环仪 1. 根据下表在冰上配制 real-time PCR 反应液 注意 : 根据反应总数计算所需的理论体积, 并额外多制备 10 % 的体积 实验中应始终设立不含 DNA 模板的阴性对照 组分 每个反应的体积 2 SYBR Green Master Mix 12.5 μl PCR Forward Primer (10 μm)* 0.5 μl PCR Reverse Primer (10 μm)* 0.5 μl Template DNA (<100 ng) # 2.0 μl ddh 2O 9.5 μl 总体积 25.0 μl * 通常引物的终浓度为 0.4 μm 是最佳的 但对于个别需要确定最佳引物浓度的实验, 可将引物从 0.1 μm 到 1 μm 进行梯度摸索 # 通常在 20 μl 反应体系中, 模板 DNA 不应超过 100 ng 由于不同模板 DNA 中的靶序列拷贝数不同, 可通过梯度稀释模板 DNA 来确定最佳用量 对于两步的 RT-PCR, 从逆转录反应中得到的未稀释的 cdna 的体积不应超过最终 PCR 体积的 10 % 2. 设置 PCR 程序 建议根据下表设置两步法 PCR 程序 如有必要, 可优化 PCR 条件 当使用 T m 值较低的引物, 难以扩增靶序列时, 可使用三步法 PCR 程序 阶段 程序 预变性 95,30 秒 PCR 变性 95, 5 秒 (40 个循环 ) 退火 / 延伸 60,30 秒 熔解曲线 3. 结果分析反应结束后, 确认 real-time PCR 的扩增曲线和熔解曲线, 对基因表达进行绝对定量时, 需制作标准曲线 17 18

12 PCR Optimization If it is not ideal for using recommended two-step PCR program, primer concentration and reaction conditions should be optimized. Accurate quantification is available in a broad range, when both specificity and amplification efficiency are met. High specificity should meet the following requirements: Primer-dimers should not be produced in NTC (No Template Control). No non-specific amplification. High amplification efficiency should meet the following requirements: Amplified targets should be detected earlier with a smaller CT (cycle threshold) value. High amplification efficiency of PCR (close to 100 %). Primer concentration optimization: The relationship between primer concentration and reaction performance is listed as follow: Reduced primer concentration could improve specificity, while increased primer concentration could improve amplification efficiency. Primer Concentration Low Concentration (0.1 μm) High Concentration (1 μm) Reaction Specificity: High Low Amplification Efficiency: Low High PCR reaction condition optimization: 1. Improve reaction specificity by increasing annealing temperature appropriately. For example: PCR 优化 如使用推荐的 PCR 程序效果不佳, 可对引物浓度和反应条件进行优化 当同时满足特异性和扩增效率时, 才能在较大范围内进行准确的定量 高特异性须满足以下要求 : 在 NTC ( 无模板对照 ) 孔没有产生引物二聚体 无非特异性扩增 高扩增效率须满足以下要求 : 扩增的靶序列能被较早地检测到,CT ( 阈值 ) 值小 PCR 扩增效率高 ( 接近 100 %) 引物浓度的优化 : 引物浓度和反应性能的关系如下 : 减少引物浓度可提高特异性, 而提高引物浓度可提高扩增效率 引物浓度 低浓度 (0.1 μm) 高浓度 (1 μm) 反应特异性 : 高 低 扩增效率 : 低 高 PCR 反应条件优化 : 1. 适当提高退火温度可提高反应特异性 例如 : Two-step PCR standard program Increase annealing temperature 2 步法 PCR 标准程序 提高退火温度 95, 5 seconds 95, 5 seconds 95,5 秒 95,5 秒 60, 30 seconds ~ 64, 30 seconds 60,30 秒 ~ 64,30 秒 19 20

13 2. Improve amplification efficiency by prolonging extension time or changing into three-step PCR program. For example: 2. 增加延伸时间或改为 3 步法 PCR 程序可提高扩增效率 例如 : Two-step PCR standard program Prolong extension time 2 步法 PCR 标准程序 增加延伸时间 95, 5 seconds 95, 5 seconds 95,5 秒 95,5 秒 60, 30 seconds 60, 60 seconds 60,30 秒 60,60 秒 Three-step PCR standard program Prolong extension time 3 步法 PCR 标准程序 增加延伸时间 95, 5 seconds 95, 5 seconds 95,5 秒 95,5 秒 55, 30 seconds 55, 30 seconds 55,30 秒 55,30 秒 72, 30 seconds 72, 60 seconds 72,30 秒 72,60 秒 3. Pre-degeneration Generally, the degeneration works well for circular plasmid hard to degenerate and genomic DNA in the condition of pre-degeneration at 95 for 30 seconds. The time of pre-degeneration could be prolonged to 1 to 2 minutes dependent on the template. However, it is not recommended to prolong the time of pre-degeneration to more than 2 minutes, due to the inactivation of enzyme. 3. 预变性通常对于难以变性的环状质粒和基因组 DNA 在 秒的预变性条件下均可很好地变性 也可根据模板状态增加预变性的时间至 1-2 分钟 但变性时间过长容易使酶失活, 不推荐将预变性时间增加至 2 分钟以上 21 22

14 Troubleshooting 常见问题与解决方案 No product, or product detected late in PCR, or only primer-dimers detected a) Annealing time too short Use the recommended annealing time. b) Extension time too short Always use the extension time specified in the protocol. 没有产物, 或荧光信号出现推迟, 或只检测到引物二聚体 a) 退火时间太短使用推荐的退火时间 c) Mg 2+ concentration not optimal d) Pipetting error or missing reagent e) PCR product too long f) Annealing temperature too high g) Primer design not optimal h) Primer concentration not optimal or primers degraded i) Problems with starting template j) Insufficient amount of starting template Always start with the Mg 2+ concentration provided. For a few targets, an increase up to 5 mm Mg 2+ may be helpful. Perform the titration in 0.5 mm steps. Check the concentrations and storage conditions of the reagents, including primers and template nucleic acids. Repeat the PCR. For optimal results, PCR products should be between 80 and 150 bp. PCR products should not exceed 300 bp. Decrease annealing temperature in 3 steps. Check for PCR products by melting curve analysis or gel electrophoresis. If no specific PCR products are detected, refer to primer design guidelines in appendix on Page 29. Use optimal primer concentrations. Refer to PCR Optimization. Check for possible degradation of the primers on a denaturing polyacrylamide gel. Check the concentration, storage conditions, and quality of the starting template. If necessary, make new serial dilutions of template nucleic acids from the stock solutions. Repeat the PCR using the new dilutions. Increase the amount of template, if possible. Ensure that sufficient copies of the target nucleic acids are present in your sample. b) 延伸时间太短 使用推荐的延伸时间 以提供的 Mg 2+ 离子浓度开始 对于某些靶序列, 将引物浓度提 c) Mg 2+ 离子浓度未优化 高至 0.5 mm 可能有帮助 以 0.5 mm 的梯度进行摸索 d) 移液出错或漏加试剂 核查试剂 ( 包括引物和模板 ) 的浓度和储存条件 重新进行 PCR e) PCR 产物太长 为达到最佳结果,PCR 产物长度应为 bp 不能超过 300 bp f) 退火温度太高 以 3 的梯度降低退火温度 g) 引物设计未优化 通过熔解曲线或凝胶电泳检测 PCR 产物 如果无特异性 PCR 产物, 参考第 30 页附录中的 引物设计原则 h) 引物浓度未优化或引物降解 使用优化的引物浓度 参考 PCR 优化 通过变性聚丙烯酰胺凝胶确认引物是否降解 i) 起始模板的问题 检查起始模板的浓度 储存条件和质量 如有必要, 将模板重新进行系列稀释, 重复 PCR j) 起始模板量不足 如有可能, 提高模板量 确保样本中的靶序列有足够的拷贝数 k) Insufficient number of cycles Increase the number of cycles. k) 循环次数不够增加循环数 23 24

15 l) No detection activated m) Wrong detection step n) Volumes of RT reaction added were are too high o) Transcript not expressed Check that fluorescence detection was activated in the cycling program. Ensure that fluorescence detection takes place during the extension step of the cycling program. High volumes of RT reaction added to the PCR may reduce amplification efficiency and the linearity of the reaction. Generally, the volume of undiluted RT reaction added should not exceed 10 % of the final PCR volume. Repeat the RT-PCR and include a positive control to make sure the absence of RT-PCR product is not due to problems with amplification and detection. l) 检测未激活 检查循环程序中荧光检测被激活 m) 检测设置错误 确保在循环程序的延伸阶段进行荧光检测 n) RT 反应物加入过多 PCR 中 RT 反应物加入过多会降低扩增效率和反应的线性 通常未稀释的 RT 反应物加入量不超过最终 PCR 反应体积的 10 % o) 转录本不表达 重复 RT-PCR, 并包括阳性对照, 以确认 RT-PCR 产物的缺失不是因为扩增和检测的原因所导致 p) Wrong detection channel/filter chosen Ensure that the correct detection channel is activated or the correct filter set is chosen for SYBR Green I. p) 选择了错误的通道 / 滤 光片 确保选择 SYBR Green I 对应的检测通道和滤光片 Primer-dimers and/or nonspecific PCR products 引物二聚体和 / 或非特异性的 PCR 产物 a) Mg 2+ concentration not optimal b) Annealing temperature too low c) Primer design not optimal d) PCR product too long e) Primers degraded Always start with the Mg 2+ concentration provided. For a few targets, an increase up to 5 mm Mg 2+ may be helpful. Perform the titration in 0.5 mm steps. Increase annealing temperature in increments of 2. Review primer design. For optimal results, PCR products should be between 80 and 150 bp. PCR products should not exceed 300 bp. Check for possible degradation of primers on a denaturing polyacrylamide gel. 以提供的 Mg 2+ 离子浓度开始 对于某些靶序列, 将引物浓度提 a) Mg 2+ 离子浓度未优化 高至 0.5 mm 可能有帮助 以 0.5 mm 的梯度进行摸索 b) 退火温度太低 以 2 的梯度升高退火温度 c) 引物未优化 重新考虑引物设计 d) PCR 产物太长 为达到最佳结果,PCR 产物长度应为 bp 不能超过 300 bp e) 引物降解 通过变性聚丙烯酰胺凝胶确认引物是否降解 f) Contamination of RNA sample with genomic DNA in RT Design primers that span exon-exon boundaries, so that only cdna targets can be amplified and detected. Alternatively, treat the RNA sample with DNase to digest the contaminating genomic DNA. f) 反转录中有基因组 DNA 污染 设计跨越外显子 - 外显子屏障的引物, 只有 cdna 靶序列可以被 扩增和检测 或者用 DNA 酶处理 RNA 样本, 去除基因组 DNA 25 26

16 No linearity in ratio of C T value/crossing point to log of the template amount a) Template amount too high Do not exceed the maximum recommended amount of template b) Template amount too lows Increase the amount of template, if possible. c) Volumes of RT High volumes of RT reaction added to the PCR may reduce reaction amplification efficiency and the linearity of the reaction. added were are too high Generally, the volume of undiluted RT reaction added should not exceed 10 % of the final PCR volume. High fluorescence in No Template control C T 值 / 模板量的 log 交叉点不呈线性 a) 模板量太高请勿超出 cdna 模板的最大推荐量 b) 模板量太低如有可能, 增加 RNA 模板的量 PCR 中 RT 反应物加入过多会降低扩增效率和反应的线性 c) RT 反应物加入过多通常未稀释的 RT 反应物加入量不超过最终 PCR 反应体积的 10 % a) Contamination of reagents b) Contamination during reaction setup Discard reaction components and repeat PCR with new reagents. Take appropriate safety precautions (e.g., use filter tips). 无模板 对照出现强荧光信号 a) 试剂污染弃去反应组分, 用新的试剂重复实验 High fluorescence in No Reverse Transcription control (RT-PCR only) a) Contaminating genomic DNA in RNA sample Varying fluorescence intensity a) Real-time cycler contaminated b) Real-time cycler no longer calibrated Design primers that span exon-exon boundaries, so that only cdna targets can be amplified and detected. Alternatively, treat the RNA sample with DNase to digest the contaminating genomic DNA. Decontaminate the real-time cycler according to the supplier s instructions. Recalibrate the real-time cycler according to the supplier s instructions. b) 反应操作中出现污染 使用适当的安全防护措施 ( 如使用带滤芯的吸头 ) 荧光强度变异 a) Real-time 仪器污染 按照厂商说明书去除 Real-time 仪器污染 b) Real-time cycler 未校准 按照厂商说明书重新校准 Real-time 仪器 27 28

17 Appendix: primer design guidelines Prerequisites for successful real-time PCR include the design of optimal primer pairs, the use of appropriate primer concentrations, and the correct storage of primer solutions. Some general guidelines are given in following table. Length of primers nucleotides For optimal results, PCR products should be between 80 and 150 bp. Length of amplified target PCR products should not exceed 300 bp. G/C content % (optimal G/C content is %) Simplified formula for estimating melting temperature (T m): T m = 2 (A+T) + 4 (G+C) T m Sequence Specificity Concentration Storage Or estimate T m value by using software (e.g. OLIGO, Primer Premier) Whenever possible, design primer pairs with similar T m values. Optimal annealing temperatures may be above or below the estimated T m. As a starting point, use an annealing temperature 5 below T m. A, C, T and G should be distribute evenly. Avoid GC rich or AT rich. Avoid a 3'-end T. G or C is better at 3 -end. Avoid complementary sequences within a primer sequence and between the primer pair. Avoid complementarity of two or three bases at the 3' ends of primer pairs to reduce primer-dimer formation. Check the specificity of primers by BLAST. Use μm of each primer in real-time PCR. For most applications, a primer concentration of 0.2 μm will be sufficient. Lyophilized primers should be dissolved in a small volume of distilled water or TE to make a 10 μm concentrated stock solution. Prepare small aliquots to avoid repeated thawing and freezing. Store all primer solutions at -20. Primer quality can be checked on a denaturing polyacrylamide gel; a single band should be seen. 附录 : 引物设计原则成功的 real-time PCR 的前提包括引物对的优化 使用合适的引物浓度以及正确的储存 以下为引物设计的一般准则 引物长度 个核苷酸扩增序列长度为达到最佳结果,PCR 产物长度应为 bp 不能超过 300 bp G/C 含量 % ( 最佳的 G/C 含量为 %) 简易估算引物退火温度的公式 :T m = 2 (A+T) + 4 (G+C) 或者使用软件如 e.g. OLIGO, Primer Premier 评估 T m T m 引物序列特异性浓度贮存 尽可能地使引物对的 T m 值接近 最佳的退火温度可能高于或低于预估的 T m 初次使用, 将退火温度设为比 T m 值低 5 A C T 和 G 应平均分布 避免某段序列富含 GC 或 AT 避免 3 端碱基为 T, 最好为 G 或 C 引物序列内及引物间避免互补序列 引物对的 3 端避免有 2 或 3 个碱基互补 通过 BLAST 检查引物的特异性 Real-time PCR 中每个引物的浓度范围为 μm 对于大部分实验,0.2 μm 的浓度已经足够 粉末状的引物可用小体积的蒸馏水或 TE 制备 10 μm 高浓度储存液 分装成小体积, 避免反复冻融, 冻存于 -20 通过变性聚丙烯酰胺凝胶检测引物的质量, 可观察到单一条带 29 30

18 Ordering Information Cat No. Product Name Size MK Trizol Total RNA Purification Kit I 50 T / 250 T MK0201 Blood Total RNA Purification Kit 50 T / 250 T MK Animal Tissue Total RNA Purification Kit 50 T / 250 T MK0203 Cultured Cells Total RNA Purification Kit 50 T / 250 T MK0204 Plant Total RNA Purification Kit 50 T / 250 T MK Bacteria Total RNA Purification Kit 50 T / 250 T MK Viral RNA Purification Kit 50 T / 250 T MK0601 cdna Reverse Transcription Kit 25 T / 100 T MK mirna Purification Kit 50 T / 250 T MK Blood/Cultured Cells DNA Purification Kit 50 T / 250 T MK Trace DNA Purification Kit 50 T / 250 T MK0101 Blood Bacteria DNA Purification Kit 50 T / 250 T MK Animal Tissues DNA Purification Kit 50 T / 250 T MK Paraffin-embedded Tissues DNA Purification Kit 50 T / 250 T MKF0101N Fast Blood DNA Purification Mini Kit 50 T / 250 T MKF0104 Fast Plant DNA Purification Kit 50 T / 250 T MKF Fast Stool DNA Purification Kit 50 T / 250 T MR bp Ladder DNA Marker 250 μl MR0102 DL2000 DNA Marker 250 μl NA0011 DNA Loading Buffer (6 ) 1 ml For more information about molecular reagents, please visit