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CONTENTS目录1General Provisions一般规定 (5)1.1Definitions定义 (5)1.2Interpretation解释 (10)1.3Communications通信交流 (11)1.4Law and Language法律和语言 (12)1.5Priority of Document文件优先次序 (12)1.6Contract Agreement合同协议书 (12)1.7Assignment权益转让 (13)1.8Care and Supply of Document文件的照管和提供 (13)1.9Confidentiality保密性 (14)1.10Employer’s Use of Contractor’s Doc uments雇主使用承包商文件 (14)1.11Contractor’s Use of Employer’s Documents承包商使用雇主文件 (15)1.12Confidential Details保密事项 (15)1.13Compliance with Laws遵守法律 (15)1.14Joint and Several Liability共同的和各自的责任 (16)2The Employer雇主 (16)2.1Right of Access to the Site现场进入权 (16)2.2Permits, Licences or Approves许可、执照或批准 (17)2.3Employer’s personnel雇主人员 (18)2.4Employer’s Financial Arrangements雇主的资金安排 (18)2.5Employer’s Claims雇主的索赔 (18)3The Employer’s Administration雇主的管理 (19)3.1The Employer’s Representative雇主代表 (19)3.2The Employer’s personnel其他雇主人员 (20)3.3Delegated Persons受托人员 (20)3.4Instructions指示 (21)3.5Determinations确定 (21)4The Contractor承包商 (21)4.1The Contractor’s General Obligations承包商的一般义务 (22)4.2Performance security履约担保 (22)4.3Contractor’s Representative承包商代表 (23)4.4Subcontractors分包商 (24)4.5Nominated Subcontractors指定的分包商 (25)4.6Co-operation合作 (25)4.7Setting out放线 (26)4.8Safety procedures安全程序 (26)4.9Quality Assurance质量保证 (27)4.10Site Data现场数据 (27)4.11Sufficiency of the Contract Price合同价格 (28)4.12Unforeseeable Difficulties不可预见的困难 (28)4.13Rights of way and Facilities道路通行权于设施 (28)4.14Avoidance of Interference避免干扰 (29)4.15Access Route进场通路 (29)4.16Transport of Goods货物运输 (30)4.17Contractor’s Equipment承包商设备 (30)4.18Protection of the Environment环境保护 (30)4.19Electricity, Water and Gas电、水和燃气 (31)4.20Employer’s Equipment and Free-Issue Material雇主设备和免费供应的材料 (31)4.21Progress Reports进度报告 (32)4.22Security of the Site现场保安 (33)4.23Contractor’s Operations on Site承包商的现场作业 (34)4.24Fossils化石 (34)5Design设计 (35)5.1General Design Obligations设计义务一般要求 (35)5.2Contractor’s Documents承包商文件 (36)5.3Contractor’s Undertaking承包商的承诺 (37)5.4Technical Standards and Regulations技术标准和法规 (37)5.5Training培训 (38)5.6As-Built Documents竣工文件 (38)5.7Operation and Maintenance Manuals操作和维修手册 (39)5.8Design Error设计错误 (39)6Staff and Labour员工 (39)6.1Engagement of Staff and Labour员工的雇用 (40)6.2Rates of Wages and Conditions of Labour工资标准和劳动条件 (40)6.3Persons in the Service of Employer为雇主服务的人员 (40)6.4Labour Laws劳动法 (40)6.5Working Hours工作时间 (41)6.6Facilities for Staff and Labour为员工提供设施 (41)6.7Health and Safety健康和安全 (41)6.8Contractor’s Superintendence承包商的监督 (42)6.9Contractor’s Personnel承包商人员 (42)6.10Records of contractor’s Personnel and Equipment承包商人员和设备的记录 (43)6.11Disorderly Conduct无序行为 (43)7Plant, Materials and Workmanship生产设备、材料和工艺 (43)7.1Manner of Execution实施方法 (43)7.2Samples样品 (44)7.3Inspection检验 (44)7.4Testing试验 (45)7.5Rejection拒收 (46)7.6Remedial Work修补工作 (46)7.7Ownership of Plant and Materials生产设备和材料的所有权 (47)7.8Royalties土地(矿区)使用费 (47)8Commencement,Delays and Suspension开工、延误和暂停 (48)8.1Commencement of Works工程的开工 (48)8.2Time for Completion竣工时间 (48)8.3Programme进度计划 (49)8.4Extension of Time for Completion竣工时间延长 (50)8.5Delays Caused by Authorities当局造成的延误 (50)8.6Rate of Progress工程进度 (51)8.7Delay Damages误期损害赔偿费 (51)8.8Suspension of Work暂时停工 (52)8.9Consequences of Suspension暂停的后果 (52)8.10Payment for Plant and Materials in Event of Suspension暂停时对生产设备和材料的付款 (53)8.11Prolonged Suspension托长的暂停 (53)8.12Resumption of Work复工 (53)9Tests on Completion竣工试验 (54)9.1Contractor’s Obligations承包商的义务 (54)9.2Delayed Tests延误的试验 (55)9.3Retesting重新试验 (55)9.4Failure to Pass Tests on Completion未能通过竣工试验 (56)10Employer’s Taking Over雇主的接收 (56)10.1Taking Over of the Works and Sections工程和分项工程的接收 (56)10.2Taking Over of Parts of the Works部分工程的接收 (57)10.3Interference with Tests on Completion对竣工试验的干扰 (57)11Defects Liability缺陷责任 (58)11.1Completion of Outstanding Work and Remedying Defects完成扫尾工作和修补缺陷 (58)11.2Cost of Remedying Defects修补缺陷的费用 (59)11.3Extension of Defects Notification Period缺陷通知期的延长 (59)11.4Failure to Remedy Defects未能修补的缺陷 (60)11.5Removal of Defective Work移出有缺陷的工程 (60)11.6Further Tests进一步试验 (61)11.7Right of Access进入权 (61)11.8Contractor to Search承包商调查 (61)11.9Performance Certificate履约证书 (62)11.10Unfulfilled Obligations未履行的义务 (62)11.11Clearance of Site现场清理 (62)12Tests after Completion竣工后试验 (63)12.1Procedure for Tests after Completion竣工后试验的程序 (63)12.2Delayed Tests延误的试验 (64)12.3Retesting重新试验 (64)12.4Failure to Pass Tests after Completion未能通过的竣工后试验 (65)13Variations and Adjustments变更和调整 (66)13.1Right to Vary变更权 (66)13.2Value Engineering价值工程 (66)13.3Variation Procedure变更程序 (66)13.4Payment in Applicable Currencies以适用货币支付 (67)13.5Provisional Sums暂列金额 (67)13.6Daywork计日工作 (68)13.7Adjustments for Changes in Legislation因法律改变的调整 (69)13.8Adjustments for Changes in Cost因成本改变的调整 (70)14Contract price and Payment合同价格和支付 (70)14.1The Contract Price合同价格 (70)14.2Advance payment预付款 (70)14.3Application for Interim Payments期中付款的申请 (72)14.4Schedule of Payments付款价格表 (73)14.5Plant and Materials intended for the Works拟用于工程的生产设备和材料 (73)14.6Interim Payments期中付款 (74)14.7Timing of Payments付款的时间安排 (74)14.8Delayed Payment延误的付款 (75)14.9Payment of Retention Money保留金支付 (75)14.10Statement at Completion施工报表 (76)14.11Application for Final Payment最终付款的申请 (76)14.12Discharge结清证明 (77)14.13Final Payment最终付款 (77)14.14Ce ssation of Employer’s Liability雇主责任的中止 (78)14.15Currencies of Payment支付的货币 (78)15Termination by Employer由雇主终止 (79)15.1Notice to Correct通知改正 (79)15.2Termination by Employer由雇主终止 (79)15.3Valuation at Date of Termination终止日期时的估价 (81)15.4Payment after Termination终止后的付款 (81)15.5Employer’s Entitlement to Termination雇主终止的权利 (82)16Suspension and termination by Contractor由承包商暂停和终止 (82)16.1Contractor’s Entitlement to Suspend Work承包商暂停工作的权利 (82)16.2Termination by Contractor由承包商终止 (83)16.3Cessation of Work and Removal of Contractor’s Equipment停止工作和承包商设备的撤离 (84)16.4Payment on Termination终止时的付款 (84)17Risk and Responsibility风险和职责 (85)17.1Indemnities保障 (85)17.2Contractor’s Care of the Works承包商对工程的照管 (86)17.3Employer’s Risks雇主的风险 (87)17.4Consequence of Employer’s Risks雇主风险的后果 (87)17.5Intellectual and Industrial Property Rights知识产权和工业产权 (88)17.6Limitation of Liability责任限度 (89)18Insurance保险 (89)18.1General Requirements for Insurances有关保险的一般要求 (89)18.2Insurance for Works and Contractor’s Equipment工程和承包商设备的保险 (91)18.3Insurance against Injury to Persons and Damage to Property人身伤害和财产损害险 (93)18.4Insurance for Contractor’s Personnel承包商人员的保险 (94)19Force Majeure不可抗力 (94)19.1Definition of Force Majeure不可抗力的定义 (95)19.2Notice of Force Majeure不可抗力的通知 (96)19.3Duty to Minimise Delay将延误减至最小的义务 (96)19.4Consequences of Force Majeure不可抗力的后果 (96)19.5Force Majeure Affecting Subcontractor不可抗力影响分包商 (97)19.6Optional Termination, Payment and Release自主选择终止、支付和解除 (97)19.7Release from Performance under the Law根据法律解除履约 (98)20Claims, Disputes and Arbitration索赔、争端和仲裁 (98)20.1Contractor’s Claims承包商的索赔 (99)20.2Appointment of the Dispute Adjudication Board争端裁决委员会的任命 (100)20.3Failure to Agreement Dispute Adjudication Board对争端裁决委员会未能取得一致时 (102)20.4Obtaining Dispute Adjudication Board’s Decision取得争端裁决委员会的决定 (102)20.5Amicable Settlement友好解决 (103)20.6Arbitration仲裁 (104)20.7Failure to Comply with Dispute Adjudication Board’s Decision未能遵守争端裁决委员会的决定 (105)20.8Expiry o f Dispute Adjudication Board’s Appointment争端裁决委员会任命期满 (105)1General Provisions一般规定1.1Definitions定义In the Conditions of Contract (“these Conditions”), which include Particular Conditions and these General Conditions, the following words and expressions shall have the meaning stated. Words indicating persons or parties include corporation and other legal entities, except where the context requires otherwise.在合同条件（“本条件”），包括专用条件和通用条件中，下列词语和措辞应具有以下所述的含义.除上下文另有要求外，文中人员或当事各方等词语包括公司和其他合法实体.1.1.1The Contract合同1.1.1.1“Contract” means the Contract Agreement, these Conditions, the Employer’s Requirement,the Tender, and further document (if any) which are listed in the Contract Agreement.“合同”系指合同协议书、本条件、雇主要求、投标书和合同协议书列出的其他文件（如果有）.1.1.1.2“Contract Agreement”means the contract agreement referred to in Sub-Clause 1.6[Contract Agreement], including any annexed memoranda.“合同协议书”系指第1.6款[合同协议书]中所述的合同协议书及所附各项备忘录.1.1.1.3“Employer’s requirements”means the document entitled employer’s requirements, asincluded in the Contract, and any additions and modifications to such document in accordance with the Contract. Such document specifies the purpose, scope, and/or design and/or other technical criteria, for the Works.“雇主要求”系指合同中包括的，题为雇主要求的文件，其中列明工程的目标、范围、和（或）设计和（或）其他技术标准，以及按合同对此项文件所作的任何补充和修改.1.1.1.4“Tender” means the Contractor’s signed offer for the Works and all other documents whichthe Contractor submitted therewith (other than these Conditions and Employer’s Requirements, if so submitted), as included in the Contract.“投标书”系指包含在合同中的由承包商提交的为完成工程签署的报价，以及随同提交的所有其他文件（本条件和雇主要求除外，如同时提交）.1.1.1.5“Performance Guarantees” and “Schedule of payments” mean the documents so named(if any), as included in the Contract.“履约保证”和“付款计划表”系指合同中包括的具有上述名称的文件（如果有）.1.1.2Parties and Persons各方和人员1.1.2.1“Party” means the Employer or the Contractor, as the context requires.“当事方（或一方）”根据上下文需要，或指雇主，或指承包商。
abi quantstudio5标准操作规程-回复Abi QuantStudio 5标准操作规程1. 引言Abi QuantStudio 5是一款先进的实时聚合酶链反应（Real-Time PCR）设备，被广泛应用于基因表达分析、突变检测、拷贝数变异分析等领域。
本文将详细介绍Abi QuantStudio 5的使用步骤和操作规范，帮助用户合理高效地进行实验。
2. 前期准备在进行实验之前，需要购买适当的试剂盒和耗材，并准备好所需的样本和试剂。
确保实验室台面整洁，工作区域干净，并根据实验所需调整设备的温度和湿度。
3. PCR实验操作步骤3.1. 设备开机准备先将Abi QuantStudio 5连接电源，并待待设备启动完成后进入“PCR”模式。
稍等片刻，直到设备温度稳定在所需温度。
3.2. 样本和试剂准备根据实验需求，将待测样本和质控样品取出，并使用合适的方法提取目标核酸。
同时，根据实验需求准备好各种试剂，包括PCR探针和引物，酶切酶等。
3.3. 样品货架设置将样品盘放在Abi QuantStudio 5的样品货架上，并按照实验需求设置样品位置。
确保每个孔位都放入正确的样品，防止交叉污染的发生。
将样品盘放置好后，轻轻轻按下样品盖，保证与孔位的完全吻合。
3.4. PCR体系配置根据试剂盒说明书，准确计量待配置的PCR体系，包括PCR反应缓冲液、模板DNA、MGCL2、引物、探针等。
使用专用的移液器和消毒的吸头进行配置，避免交叉污染。
3.5. PCR程序设置在QuantStudio软件中选择适当的PCR模式，并根据试剂盒说明书输入反应体系和实验方案参数。
确保PCR程序模板的准确性和完整性，避免因设置错误导致结果不准确。
3.6. PCR实验运行将配置好的PCR体系添加到样品盘对应的孔位中，注意不要发生溢液现象。
关闭样品盖，检查无异常后，将样品盘放入设备，并点击软件上的“Run”按钮开始PCR实验。
实验过程中，根据实验需求进行必要的参数调整。
PCR-ELISA端粒酶检测法发布日期:2007-6-27 热门指数:782 收藏端粒是真核生物染色体末端的特异DNA-蛋白结构，端粒DNA是一系列重复的富含G的DNA序列，这一序列在生物进化中有高度的保守性（人重复序列为TTAGGG）。
已确认端粒在保护基因组DNA不被降解、防止染色体有害的结合（如染色体末端融合、重排、染色体移位和染色体缺失）中起重要作用。
由于DNA聚合酶不能复制线性DNA的最末端，多数正常体细胞的端粒末端每经一个复制循环就进行性缩短。
这一现象在体内和体外都得到证实，并且可能与高级真核生物的正常体细胞有限的繁殖能力有关。
这一活性可能在与细胞衰老有关的情况中起作用。
与体细胞相对照，生殖细胞是永生性的，它需要为将来器官形成保存全部的遗传信息。
因此它必须对抗端粒缩短。
这一工作通过在基因组染色体的末端添加新的端粒重复序列而完成。
端粒酶是一种核糖核蛋白，它们用自身的RNA成份的互补序列作模板，催化TTAGGG 重复序列加到染色体末端。
在大多数永生性细胞株和肿瘤细胞株中也可见端粒酶活性的表达。
端粒酶反应超越了细胞的增殖限制，这可能是恶性肿瘤发生的一个先决条件。
传统的端粒酶研究方法是以探针延伸为基础测定端粒酶活性。
由于需要大量的样品材料，且检验灵敏度受局限, 这一方法已被端粒重复扩增法（Telomeric Repeat Amplification Protocol, TRAP）所取代。
标准的TRAP测定是通过PCR扩增端粒酶反应的产物，使用放射性标记，经凝胶电泳后，通过放射自显影显示结果。
这里介绍使用非放射性标记的检测端粒酶的新方法：活性光密度酶联免疫测定法。
这种方法具有如下特点：安全，无需使用放射性同位素一步反应，使用即用的混合物使端粒酶介导的引物延伸和PCR扩增可在一个试管中进行。
应用广泛，扩增后可适用于不同分析法。
灵敏，与放射性同位素TRAP测定相当。
可靠，准确、重复性好。
快速，6-8小时得到结果。
LightCycler® 480 中文操作说明罗氏医学仪器公司 / 应用科学部门February 2008目录一. 启动机器与软件 3二. 软件设定 --- 开启新实验 5三. 样品编辑9四. 结果分析12五. 报告18六. 数据转移至其它计算机(数据输出与输19 入)七. 使用者管理19一．启动机器与软件•启动机器1.机器主开关位于机器右后方。
2.机器正面两个警示灯代表机器状态。
待警示灯 (右) 由闪烁橘色灯转变成稳定橘色灯，警示灯 (左) 由橘色转变成绿色 (约三分半钟)，表示机器已经启动完成。
3.按压机器正面上唯一的按钮，放入已经封膜完成之 96 或 384 孔盘。
放置完成后，左右两警示灯都呈现绿色。
•启动软件1.开启计算机，并以正确的使用者名称与密码登入 Windows。
User name: OPERATORPassword: LC4802.开启 LightCycler 480 软件，并输入正确的使用者与密码。
二．软件设定-- 开启新实验•设定新实验1.软件开启后，会进入主画面。
点选“New Experiment”。
2.出现实验设定窗口。
任何时候需要回主画面的话，点选画面右边的按钮。
•设定实验❖若要套用设定好的实验步骤 (Template)，请跳至 6.。
1.在实验设定画面上方，先选择适当的 Detection Forma t。
选项包含:另外可再点选“Customize” 勾选所需的滤片组合。
2.输入反应体积: 96 well plate 范围为 10-100 ul，而 384 well plate 范围为 5-20 ul。
3.设定实验步骤 (Program)•设定 program，包含有 Pre-incubation, Amplification (PCR), Melting curve (optional) 与 Cooling。
利用『+』与『－』增加或删除步骤。
•设定循环数目 (Cycle) 与分析模式。
Hinged frame 【铰练式枪⾝】HMG (Heavy Machine Gun) 【重机枪】Hold-open device 【枪栓固定卡榫】Hollowpoint 【空尖弹头】Holster 【枪套】Hopper feed 【漏⽃给弹】Hot load 【加码剂量】Ignition 【引燃】Inertia firing pin 【惯性撞针】Interchangeable barrel 【可更换枪管】International Clay Pigeon 【国际不定向飞靶】International Skeet 【国际定向飞靶】International Trap 【国际不定向飞靶】IPSC 【国际实⽤射击联盟】Iron sight 【⾦属瞄准具】Isosceles 【等腰式射姿】Jacket 【被覆层】Jag 【擦枪头】JHP(Jacket Hollow Point) 【空尖⾦属被覆弹】JSP(Jacket Soft Point) 【软头⾦属被覆弹】Jump 【枪⼝上扬】Kentucky Rifle 【肯塔基来福枪】Keyhole 【钥孔形弹孔】Kick 【后座感】Kurz 【短】Laminated 【复合夹板】Land 【阳膛】Lane 【靶道】Laser sight 【雷射瞄准具】Lead 【铅】读⾳"led"，⽤来制造弹头或弹头核的软质⾦属，通常会混合⼀点其他⾦属以增加硬度。
Lead 【膛喉】Lead fouling 【铅垢】Lead shaving 【削弹头】Lever action 【杠杆式枪机】LMG (Light Machine-Gun) 【轻机枪】Load 【装药剂量】Loaded chamber indicator 【上膛显⽰杆】Loading gate 【装弹⼝盖】Loading port 【装弹⼝】Loading press 【装弹压床】Lock 【枪机】Lock time 【闭锁时间】Locking flaps 【闭锁活板】Locking lug 【闭锁凸⽿】Long action 【长枪机】Long gun 【长枪】Long recoil 【长后座⾏程】Long stroke 【长⽓体活塞⾏程】LSW (Light Support Weapon) 【轻型⽀援武器】Lug 【凸⽿】Lug 【凸杆】Machine gun 【机枪】Machine Pistol 【全⾃动⼿枪】Magazine 【弹匣】Magazine 【弹仓】Magazine cutoff 【弹仓隔断器】Magazine pouch 【弹匣袋】Magazine release 【弹匣卡榫】Magazine safety 【弹匣保险】Magnum 【麦格农】Mainspring 【击发簧、击锤簧】Mannlicher 【曼⾥契】Mannlicher stock 【曼⾥契护⽊】Manual safety 【⼿动保险】Marksman 【神射⼿】Master eye 【优势眼】Match 【射击竞赛】Matchlock 【⽕绳枪】Matt blue 【钝蓝】Matt stainless steel 【钝不锈钢】Mauser rifle 【⽑瑟枪】Maxim gun 【马克沁机枪】Meplat 【弹尖】Metallic sights 【⾦属照门】Mil-Dot 【毫弧度点】Milled 【铣削】⽤机器加⼯切削成形。
美国应用生物系统公司ABI Prism® 7000型荧光定量PCR仪操作手册快速入门指南美国应用生物系统中国公司· 技术服务部2003年ABI Prism剖7000中文操作手册目录一. 开机 (3)二. 实时定量的软件运行 (3)1. 新建文件 (3)2. 探针设置 (4)3. 填样品表 (4)4. 参比荧光 (5)5. 循环参数 (5)6. 启动扩增 (5)三. 实时定量的数据分析 (6)1. 数据分析 (6)2. 基线设定 (6)3. 线性图谱 (6)4. 原始数据 (7)5. 标准曲线 (7)6. 实验报告 (7)四. 终点读板的软件运行 (7)1. 新建文件 (8)2. 探针(D ETECTOR)设置 (8)3. 位点(M ARKER)设置 (8)4. 填样品表 (8)5. 参比荧光 (8)6. 终点读板 (9)五. 终点读板的数据分析 (9)1. 数据分析 (9)2. 信号分布 (9)3. 基因分型 (9)4. 保存结果 (9)六. 日常维护 (10)1. 荧光污染的检查与处理 (10)2. 检测器光源的更换流程 (10)2ABI Prism剖7000中文操作手册ABI Prism剖7000型荧光定量PCR仪快速入门指南一. 开机1. 确认电脑与主机的数据通讯线(灰色USB连线)连接正确。
2. 确认电脑处于外接电源供电状态。
3. 启动电脑，以Administrator用户名登录，进入Windows2000操作系统。
4. 待桌面图标出现后，打开7000主机的电源。
5. 待主机的电源指示灯点亮后，启动7000 SDS应用软件。
在进行实验之前，请先检查样品加热模块以确定它没有受到荧光污染，具体方法请按照第6节的“荧光污染的检查与处理”流程进行。
二. 实时定量的软件运行基因表达的绝对定量和相对定量研究、转基因食品的检测(GMO)、各种病原体的检验检疫等各种定量应用；以C T值的大小作为样品阴性、阳性判定依据的定性应用；以及SNP 检测、等位基因鉴定实验等的第一步PCR扩增，都是采用实时定量模式，按照以下步骤操作。
realtime pcr操作流程英文回答：Real-time PCR, also known as quantitative PCR (qPCR), is a powerful molecular biology technique used to amplify and quantify specific DNA sequences in real-time. It is widely used in various fields, including gene expression analysis, pathogen detection, and genetic testing. The workflow of a real-time PCR experiment typically consists of several key steps.1. Experimental Design:Before starting a real-time PCR experiment, it is essential to design the experiment carefully. This includes selecting the target DNA sequence to be amplified, designing specific primers and probes, and optimizing reaction conditions.2. Sample Preparation:The next step involves preparing the DNA samples for analysis. This can include extracting DNA from cells or tissues, purifying the DNA, and quantifying its concentration using a spectrophotometer or fluorometer.3. Primer and Probe Design:Specific primers and probes are designed to target the desired DNA sequence. Primers are short DNA sequences that bind to the complementary regions of the target DNA, while probes are labeled with fluorescent dyes and bind to the amplified DNA during the PCR reaction.4. PCR Reaction Setup:The PCR reaction mixture is prepared by combining the DNA template, primers, probes, and other components such as buffer, dNTPs, and DNA polymerase. The reaction mixture is then loaded into individual wells of a real-time PCR plate.5. Thermal Cycling:The real-time PCR instrument performs a series of temperature cycles to amplify the DNA. The thermal cycling typically consists of three steps: denaturation, annealing, and extension. The denaturation step separates the DNA strands, the annealing step allows the primers to bind to the target DNA, and the extension step synthesizes new DNA strands using DNA polymerase.6. Fluorescence Detection:During the PCR cycles, the real-time PCR instrument monitors the fluorescence emitted by the probes as they bind to the amplified DNA. The increase in fluorescence intensity is directly proportional to the amount of DNA amplified in each cycle.7. Data Analysis:The real-time PCR instrument continuously measures the fluorescence intensity and generates amplification curves. These curves represent the increase in DNA concentrationover the PCR cycles. The data can be analyzed using various software programs to determine the initial DNA concentration, quantify gene expression levels, or detectthe presence of specific pathogens.中文回答：实时荧光定量PCR（real-time PCR），也称为定量PCR （qPCR），是一种用于实时扩增和定量特定DNA序列的强大的分子生物学技术。
realtime pcr操作流程英文回答：Real-time PCR, also known as quantitative PCR (qPCR), is a widely used technique in molecular biology to detect and quantify DNA or RNA molecules. It is a sensitive and accurate method that allows researchers to measure gene expression levels, identify genetic variations, and detect pathogens.The general workflow of a real-time PCR experiment involves several steps:1. Sample preparation: This step involves extracting DNA or RNA from the sample of interest. Various methods can be used for sample preparation, such as phenol-chloroform extraction, column-based purification kits, or automated extraction systems. The quality and purity of the extracted nucleic acids are crucial for the success of the PCR reaction.2. Primer design: Primers are short DNA sequences that specifically bind to the target DNA or RNA region of interest. They are designed using bioinformatics tools and should have high specificity and minimal secondary structures. The forward and reverse primers are designed to flank the target sequence, and ideally, they should have similar melting temperatures.3. Reaction setup: The PCR reaction mixture is prepared by combining the extracted nucleic acids, primers, a DNA polymerase enzyme, and a fluorescent dye or probe. The reaction mixture is typically prepared in a PCR tube or plate. It is important to maintain proper aseptic techniques to prevent contamination.4. Thermal cycling: The PCR reaction goes through a series of temperature cycles in a thermal cycler machine. These cycles typically include denaturation, annealing, and extension steps. The denaturation step separates the DNA double strands, the annealing step allows the primers to bind to the target sequence, and the extension stepsynthesizes new DNA strands using the primers as templates. The number of cycles depends on the initial amount oftarget nucleic acid and the desired level of amplification.5. Data collection and analysis: Real-time PCR instruments monitor the fluorescence emitted by the fluorescent dye or probe during each cycle. The fluorescence signal increases proportionally to the amount of amplified DNA or RNA. The data can be plotted in a graph called the amplification curve, which shows the exponential growth of the target sequence. The threshold cycle (Ct) value, which represents the cycle number at which the fluorescence signal crosses a predetermined threshold, is used to quantify the initial amount of target nucleic acid.Real-time PCR is a versatile technique that can be adapted to various applications, including gene expression analysis, pathogen detection, and genetic testing. Itoffers high sensitivity, specificity, and quantification accuracy, making it an essential tool in molecular biology research and diagnostic laboratories.中文回答：实时荧光定量PCR（real-time PCR），也被称为定量PCR （qPCR），是分子生物学中广泛使用的一种技术，用于检测和定量DNA或RNA分子。
Real-time PCR（实时聚合酶链反应）是一种用于检测和定量DNA或RNA的技术。
它可以在PCR（聚合酶链反应）进行的实时监测PCR 产物的累积量。
real-time PCR技术适用于病毒检测、基因表达分析、SNP检测等许多领域。
real-time PCR技术的操作流程相对简单，但需要一定的实验操作技巧和严格的操作规范。
以下是real-time PCR的简易操作流程：1. 样品准备- 从实验对象中收集所需的组织样品或细胞样品。
- 样本的处理和提取要遵循标准的生物安全操作规程，确保样品的纯度和完整性。
2. DNA/RNA提取- 根据实验需求选择合适的DNA/RNA提取试剂盒和方法，从样品中提取目标DNA或RNA。
- 保证提取的DNA/RNA质量和浓度满足real-time PCR的要求。
3. Primer和探针设计- 根据目标基因序列设计引物和探针，确保其特异性和有效性。
- 引物和探针的设计应遵循一定的原则和标准，可以使用专业的设计软件或委托专业实验室进行设计。
4. PCR体系设置- 根据实验设计和引物/探针的特性设置PCR反应体系，包括引物和探针的最佳浓度、PCR反应缓冲液、酶和模板DNA/RNA的加入量等。
- 严格按照实验方案中的要求和比例进行PCR反应体系的设置，确保反应的准确性和稳定性。
5. PCR反应- 将PCR反应体系加入到real-time PCR仪器中，根据实验方案设置好PCR程序。
- 在PCR过程中，实时监测PCR产物的累积量，记录和分析反应曲线。
6. 数据分析- 根据实验目的和方法选择合适的real-time PCR数据分析软件，对实验数据进行处理和分析。
- 分析数据并计算目标基因的相对表达量或浓度，进行统计学分析和结果解读。
7. 结果呈现- 将实验结果以图表或表格的形式清晰呈现出来，配以必要的文字解释。
- 结果的呈现应简洁明了，突出实验的关键发现和结论。
real-time PCR技术的操作流程虽然相对简单，但在实际操作中仍然需要严格遵循操作规范和注意实验细节。
ORIGINAL ARTICLEProbe - Animal Science Volume 1 Issue 1 | 2019 | 16REplication jumping duration PCRAmplification,Closing and Sanger sequencing of a fragment Of the temptation gene in black-spotted frog1Xindi CuiInner Mongol University of Technology,Shijiazhuang,Hebei,ProvinceAbstract:During in vitro replication of DNA by DNA Polymerase,The diverse palindromic StructuresOn templates of DNA can lead to replication jumping and cause fragments to be missing from daughter strands.We recorded replication jumping in which a 564bp fragment was missing from daughter DNA.The original template was a 1126bp fragment of the introduction of the temporary gene of black-spotted frog(Pelophylax nigromaculatus).The replication jump was recorded after amplifica-tion of this template using polymerase chainREaction,Closing Using Vector,And sequencing using the Sanger Method.The replication jump was elevated to rise from this series of events:Keywords:Reloading DNA polymerase;Replication;vitro processes;fragment;replication jumpPolymerase Chain Reaction(Polymerase ChainREactionPCR)Is refers to inDNAPolymerase of catalytic under to mother chainDNAFor template to specific primers for extension starting point by degeneration,Annealing,Extension and steps in vitro copy the and mother chain TemplateDNAComplementary of sub-chain DNAOf process is a can fast,Specifically in vitro amplification objectiveDNAOf technology is biological research in application the most widely[2-4]Laboratory Technology one of PCRProduct often need to After cloning after the sequencing or other Analysis.Cloning an arcane willPCRProduct and specific carrierDNAMolecular connection formation recombinantDNAMolecular into competent cells in by cells and Carrier of emerging suchT-Carrier and in very big degree on the simplified.PCRProduct of cloning process at the same time also improve the cloning Effect [8-10]Rate But,PCRNot always completely faithful of In addition to primers and template of non-specific binding was to produce non-specific opposite sex amplification product outside because template of superior structure,Polymerase of some properties such as also often will mediated-DNAOf recombinant(Recombination),Copy sliding(Slippage)AndRecombinant isDNAPolymerase in In accordance with template synthesis new chain of when because the other a and template sequence height similar of fragment and template in space on the close,polymerase from original of template chain on the move to the fragment on the continue to synthesis New Chain Final makePCRProduct become two different template chain[12]The recombinant product.Copy sliding in microsatellite and short tandem repeat sequence of amplification in very common,DNAPolymerase in synthesis new chain more.A or a few repeat unit causedPCRProduct than template chain shorten[13].Repeat unit integer times Jump amplification is when templateDNATheir own because the original sequence formation more stability of ring tongue local secondary structure New Chain Synthesis whenDNAPolymerase move to theCopyright © 2019 .This is an open-access article distributed under the terms of the Creative Commons Attribution Unported License (/licenses/by-nc/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.regional after it can span this ring tongue area continue to down synthesis new chain to makePCRProduct is template lost theAmplification people18S rDNAGene when found the middle missing54BPOf abnormal.Is big fragment of jump more difficult to happen in experimental also very rare.Because so,PCROf jump problem often be ignored caused error results.PCRProduct of cloning is use bacteria inDNAPolymerase and copy system will targetDNAFragment the in body copy.Because bacteria Copy System of faithful of much higher than in generalTaq DNAPolymerase(No3'-5'Enzyme Activity)Molecular cloning using plasmid replication is generally considered to be very accurate..However,in special cases,such as when the target molecule contains some special palindrome structure,the bacterial plasmid.Jumps or slides during replicatio.We're studying the black-spotted frog.(Pelophylax nigromacula-Tus)Antimicrobial PeptidesTemporinWhen it comes to gene structurePCRAmplification and amplification productsT-AJumps occur simultaneously during cloning and cause upBPThe fragment is missing and found with a segment6BPAbout the repeat unit.We reported the results of this experiment to provide a reference for more research..1.Materials and Methods1.1SampleChuan,Xi'an,Taiyuan,etc.20Adult black spotted lateral pleats collected in different regions,and each region5.Only, the hind leg muscle samples were taken immediately after double-damaged pulp was executed in the wild and preserved in liquid nitrogen,and then transferred-80Bao in the refrigerator Save.1.2GeneralDNAExtraction20mgAdd muscle tissue after being cut25μL protein-ase K(10mg/ml),35μL SDS(10%),295μL tne Buffer(PH=8), Fully mix and then centrifuge,56Water bath digestionH.Digestive JuiceAxyprepGenomeDNAExtraction kit(Axyprep,Hangzhou)Total ExtractionDNA,Operation in accordance with the instructions.As shown in figure1PCRProduct by agarose electrophoresis found big about73%Of individual in1200BPAnd550 BPThe at the same time appear clear of two,18%Individual short of strip signal weaker,9%The individual only1200 BPOf Strip.Will two the bands respectively recovery after and sequencing results display is long of bands1126BPShort of bands562BPAfter is in the former198bpThe start missing564bpMade.ference for two different of allele gene.But in the long fragmentSangerSequencing when found a kind of abnormal phenomenon template actual length1126BPBut sequencing results in sometimes will only get562BPOf Short Sequence.The short sequence is long fragment in BPThe start missing564bpMade.This show that short very may isPCRCause of a kind of illusion the in amplification reaction in the fragment missing.With recovery of long fragment as an Template PCRAmplification agarose gel electrophoresis detection PCRProduct when found3A situation A is only get1126BPThe long fragment A is only get562BPOf Short Fur ther more two fragment at the same time(Figure2)OnPCRProduct sequencing also prove that the this a little.Template pollution of possibility we the long fragment purification recovery after.T-ACloning.Extraction positive cloning of plasmid after with restriction enzymePst IAndNot IToPCRProduct from carrier on cut down again by agarose electrophoresis detection found positive cloning in containing two kind of cloning fragment A is containing and long fragment completely same of cloning Fragment,A kind of IS and short completely same of cloning fragment(figure 3).That is,1126BPLong of objective fragment in different colony in become size two of Fragment.Sequencing show that short remain is long fragment198bpThe missing564bpMade.This show that this1126BP Long of fragment not only inPCRAmplification process in can happen missing and in Escherichia coli of in of plasmid copy process also can Probe-Animal Science Volume1Issue1|2019 |17occur missing this a kind of phenomenon is very rare.Use softwareREpeataround2.1Okay1126BPOf fragment the repeat sequence analysis results display the sequence has the same direction repeat sequence(Direct repeats)70Of,Reverse repeat sequence(Inverse re-peats)13Of,Mirror repeat sequence(Mirror repeats)21Of and complementary repeat sequence(Complementary repeats)21Of(Table1). Will1126BPAnd526BPThe sequence of the two fragments was compared and found that the two sequences at the upstream and end of the missing fragment have one6BPSame-direction repeat sequence5'-AGACCT-3'(Figure4.)Is where the sequence deletion occurred,so far we speculateDNAA jump occurred while copying to the second repeat sequence(PCRJumping).2.DiscussionDNAA sequence can be repeated in the same direction,in the same direction[20]Jump or slide.In this study1126 BPDetected in Fragment Different repetitive sequences(Table1.),But repeated amplification,only in194~199 BPLocation and first757~762BPRepeat sequence that appears on the location5'-AGACCT-3'A jump occurred at the site, resulting in the deletion of the amplified product564bp.Surprisingly,by this pair of repetitions The jump will occur in vitro and in vivo[21.]CanceillTwo replication-hopping models are proposed..One is related to the double strand hairpin structure of the parent..The strand synthesis is closed at the base that forms the hairpin structure,and then throughDNAPolymerase Chain replacement activity continued and finally slid over the hairpin Structure.However,the parental double helix model does not seem to be applicable to our replication Jump Events because in our study,a repeat sequence is missing in the sub chain.This indicates that one of the repeat sequences is incorporated into the hairpin structure, Yes,in our study,the sequence after the first repeat or before the second repeat cannot form a stable hairpin structure with the repeat sequence(Figure5AAnd5b).The other model is the heterohelix model,which blocks the synthesis of the chain at the first repeat, whileDNAPolymerase and sub-chain are detached from the template.In the next cycle,the new chain acts as a complementary chain,matching the second repeat unit.,Annealed and became primers,DNAPolymerase rebinds, starting downstream of the second repeat unit to complete the new chainBecause in this case a repeat sequence is missing in the sub chain,which is consistent with our case..The only difference between our results and the model is the weak double chain body at the bottom of the hairpin.It is reported that,DNAPolymerase shedding and chain extension blocking may not be caused by hairpin,butDNA This structure appears when the copy occurs,which leads to the occurrence of jumps(Figure5C).We were unable to test this prediction further,but we speculated that both high-level structural-induced strand synthesis blocking and Polymerase shedding were associated with repeat sequences,and this happens both in vivo and in vitro..DNAThe ability of Polymerase to produce jumping is related to its chain replacement activity.When plasmid replication is done in bacteria,DNAPolymeraseIII(Early replication,sometimesDNAPolymeraseI)[24]When making a new chain extension,because of its chain[25],Replacement activity is not highAppropriate advanced knot on encountered Template After shedding,the new chain can be reannealed with another homologous sequence(the second repeat unit in this study,and finish the chain extension and jump.Single clone in this studyDNAThis is probably the case when the fragment is cloned again.(Figure5C)In factPCR,Sanger Sequenced or Molecular Cloned DNA The type of PolymeraseTaq DNA Poly merase,DNA Poly meraseII,T4Polymerase,etc.A high structure that is easy to jump and is also prone to replication jumps when these enzymes are involved in replication.The replication jump encountered by this research not only happens inTemporinThe two-way amplification of the gene intron also occurs inSangerIn the process of sequencing,it even happened in the Process of plasmid replication in bacteria..Based on our confirmation experiment,the purified1126BPThe fragment was used as a template for Probe-Animal Science Volume1Issue1|2019 |18reamplification and the positive plasmid was digested with double enzymes,which led to this conclusion;otherwise, jumping may be misunderstood as an independent allele..So,we 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