ap12_macroeconomics_scoring_guidelines
美国FDA生物制品无菌试验规程
生物制品无菌试验规程（WHO 1995修订讨论稿）前言《生物制品无菌试验规程》自1973年发表后又有了一些新进展，因此，将检查支原体的无菌试验要求修订如下：5.3检查支原体的无菌试验（ WHO TRS和 No．530，1973，Annex 4）该节以下文取代：检查支原体采用琼脂和肉汤培养基培养法或指示细胞培养DNA 染色法。
在要求基础细胞库、生产用细胞库、病毒种子批（或库）、或对照细胞进行支原体检查时，采用培养法和指示细胞培养法。
在要求病毒收获液、疫苗原液或成品批进行支原体检查时，采用培养法。
必要时，亦可用指示细胞培养法筛选培养基。
培养法和指示细胞培养法见附录。
其他方法，只要列出详细步骤，井用本法验证过，亦可采用。
附录检查支原体培养法和指示细胞培养法1．培养法1．1培养基的选择试验采用足量固体和液体培养基，以确保在选定的培养条件下检出产品中可能存在的少量支原体。
液体培养基应含酚红。
培养基的营养性能最少应用下列支原体验证：口腔支原体（M．orale）（人用疫苗）；肺炎支原体（M．pneumoniae）（人用疫苗）；猪鼻支原体（M．hyorhinis）（非禽类兽用疫苗，例如 DBS 1050）；鸡败血支原体（M．gallisepticum）和滑液支原体（M. Synoviae）（在生产过程中采用禽类材料或疫苗拟供家禽使用）。
采用低代试验菌株，冻存或冻干保存。
菌种经克隆后，应以适当方法确认是规定的菌种。
有些国家进行嵌入支原体检查。
1·2培养条件将已接种的培养基分成两份，一份置需氧条件下培养，另一份置厌氧条件下培养。
需氧条件是在含5—10％CO2和适当湿度的空气环境中培养。
厌氧条件是在含5－10%CO2和适当湿度的氮气环境中培养。
1．3营养性能用适宜的试验菌接种选定的培养基，每支固体培养基干皿不少于200CFU，不超过400CFU，每支液体培养基容器不少于20CFU，不超过40CFU。
每种菌株单独接种一支平皿和液体培养基。
CLIACAP 二代测序临床检测标准操作指南
CLIA/CAP 二代测序临床检测标准操作指南2015-04-23自由度本临床二代测序操作指南来自于Clinical Laboratory Improvement Amendments (CLIA) of 1988/College of American Pathologists (CAP) laboratory。
释义：Clinical Laboratory Improvement Amendments (CLIA) of 1988：美国临床实验室改进修正法规’88 。
这个法规里面有关于医学检验项目的一些质量规范，中国的临床行业很多项目的指标都参考过CLIA’88 中的内容。
College of American Pathologists (CAP) laboratory：美国病理学家协会。
相较于Sanger测序，二代测序技术（新一代测序技术，NGS）拥有高通量和低单碱基成本的特点，这使其在临床检测领域拥有得天独厚的优势。
过去的数年间，相当数量的NGS服务商迅速成长，而美国病理学家协会却没有及时依照美国临床实验室改进修正法规（CLIA）编制指南来规范这个检测。
基于NGS平台的临床检测是一种新的检测方式，相较于原有检测方法，其复杂程度大大提高，因此迫切需要一个规范标准来指导各实验室进行检测。
CAP 于2011年成立了NGS工作部门，商讨NGS临床检测实验检查表的内容。
本次CAP列出共计18条实验室要求的检查表，包含检测的二代测序实验过程和生物信息学分析过程。
二代测序技术（NGS）包含两个部分：实验操作部分（wet bench）和生物信息分析部分（dry bench）。
实验操作部分包括以下部分或所有的流程：病例样本的采集处理、核酸提取、片段化、生物分子标签（barcode）、目标区段/基因捕获富集、连接、扩增、文库构建、上机测序、数据产出。
生物信息分析部分主要依赖于高性能的计算机和不同的生物信息分析方法，主要流程包括：参考序列比对、组装、变异检测、注释、临床解释（依赖于是否运用诊断工具）。
微生物基因组学
年份 Year
基因组学文章 Articles about Genomics
基因组学综述 Reviews about Genomics
转录组学文章 Articles about Transcriptomics
转录组学综述 Reviews about Transcriptomics
2000
微生物基因组的特点
类别
特征
染色体结构
多为一条环状闭合双链DNA
基因组大小
从0.16-13Mb
编码序列
占基因组总长度的90%，平均为1Kb左 右
GC含量
16.6%-74.9%
DNA链组成的非 GCskew、ATskew、基因方向性偏好、
对称分布
密码子使用偏好
Circular representation of the genome of T. tengcongensis MB4
2010
8986
1487
296
70
截至2011年4月24日，NCBI上记录了1534个细菌基因组, 包括了103个古细菌和1431个真细菌, 其中中国科学家 完成了44个
研究现况及内容
细菌
研究内容
病原菌
毒力因子、致病岛、 耐药基因、耐药机制 以及与寄主的关系等
极端环境 极端环境下的生存机
生长的细 制，如嗜热菌的热稳
tRNAs rRNA operons
Protein with known or predicted fuction
Conserved hypothetical proteins Hypothetical proteins
2171616 41.62
932 2059
外胚层发育不良受体EDA2R的研究进展
肿瘤坏死因子受体超级家族（tumor necrosis fac⁃tor receptor superfamily，TNFRSF）的死亡受体（death receptor）以及它们的配体在胚胎正常发育及机体免疫和炎症反应过程中扮演了重要角色。
外胚层发育不良受体（ectodysplasin A2receptor，EDA2R）是一个在20年前被鉴定出来的TNFRSF成员（TNFRSF27）［1］，在肿瘤发生、雄激素性脱发等过程中起到重要的作用，但对于该受体作系统性介绍的综述文章尚未见报道。
本文就该受体的研究进展作一系统性的综述，旨在为相关研究提供新的思路。
1EDA2R的蛋白结构和配体1.1EDA2R的蛋白结构EDA2R基因位于人类染色体Xq12，全长约43kb，有6个外显子（GenBank登录号：NG_013271），外胚层发育不良受体EDA2R的研究进展蓝希钳1，2，肖海婷1，2，罗怀容1，2，陈建宁1，2（西南医科大学药学院：1衰老与再生医学实验室，2药理学教研室，四川泸州646000）【摘要】外胚层发育不良受体EDA2R（ectodysplasin A2receptor）是肿瘤坏死因子受体超级家族（tumor necrosis factor recep⁃tor superfamily，TNFRSF）中的一个较新的成员，在发育中的胚胎里有很高的表达，在成年人和动物的多个器官组织中也有表达。
与其它TNFRSF成员不同，尽管EDA2R蛋白在胞内没有死亡结构域（death domain，DD），但它仍可激活NF-κB和JNK通路，并介导细胞的凋亡。
本文广泛回顾了近年来与EDA2R有关的文献，就该蛋白分子的相关研究进展进行综述，以期为与该蛋白相关的分子功能或其介导的相关疾病的研究提供新的思路。
【关键词】EDA2R受体肿瘤坏死因子受体超级家族死亡结构域凋亡【中图分类号】R34文献标志码A doi：10.3969/j.issn.2096-3351.2021.03.018Research progress of ectodysplasin A2receptorLAN Xi-qian1，2，XIAO Hai-ting1，2，LUO Huai-rong1，2，CHEN Jian-ning1，2 1Key Laboratory for Aging and Regenerative Medicine；2Department of Pharmacology，School of Pharmac，South⁃west Medical University，Luzhou646000，Sichuan，China【Abstract】Ectodysplasin A2receptor（EDA2R）is a relatively new member of the tumor necrosis factor re⁃ceptor superfamily（TNFRSF），and it is highly expressed in developing embryos and is also expressed in multiple organs and tissues of adult human and animals.Different from other TNFRSF members，EDA2R protein does not contain the death domain in the intracellular region，but it can still activate the NF-κB and JNK pathways and medi⁃ate cell apoptosis.This article reviews related articles on EDA2R in recent years and related research advances in this protein，in order to provide new ideas for research on molecular functions associated with EDA2R or related dis⁃eases mediated by EDA2R.【Key words】Ectodysplasin A2receptor Tumor necrosis factor receptor superfamily Death domain Apoptosis基金项目：泸州市科技局－西南医科大学联合项目（2018LZXNYD-ZK12）；西南医科大学-泸州市中医医院基地项目（2019-LH005）第一作者简介：蓝希钳，博士。
产品信息和操作指南
产品信息和操作指南Human CCL17/TARC Precoated ELISA kitCat# : 1117542本试剂盒专用于科研，而非用于诊断HumanCCL17/TARC1117542目录产品简介 (1)知识背景 (1)试剂盒组分 (2)需要实验者自行准备的试剂与仪器 (2)注意事项 (3)试剂的配制 (5)操作过程 (7)结果分析 (9)试剂盒的保存 (9)操作步骤一览表 (10)参考文献 (11)ELISA测定中可能出现的问题及解决方法 (12)达优®ELISA系列产品 (15)1、产品简介：达优®人CCL17/TARC ELISA试剂盒是通过酶联免疫吸附技术，体外定量检测人血清、血浆、缓冲液或细胞培养液中的CCL17，可同时检测天然的和重组的CCL17。
本试剂盒为预包被板，整个过程孵育时间不超过5小时，洗涤9次。
本试剂盒专用于科研，而非用于诊断。
使用前请仔细阅读说明书并检查试剂盒组分，若有任何疑问请与深圳市达科为生物工程有限公司联系，E-mail：RD@.检测范围：500-7.8 pg/mL灵敏度：5 pg/mL重复性：板内变异系数＜10％,板间变异系数＜15％。
2、知识背景CCL17，又称为胸腺和活化调节趋化因子（thymus and activation regulated che-mokine ，TARC），分子量在7～12 kDa。
CCL 17产生于树突状细胞、内皮细胞、角质形成细胞和成纤维细胞，在胸腺中表达，但仅在植物血凝素刺激的外周血单个核细胞中瞬时表达。
身体其他部位如皮肤、粘膜、腺体上一些上皮来源的细胞也能合成和分泌CCL17，如肺、结肠、小肠、支气管上皮细胞等。
近年来有关研究发现，CCL17／CCR4不仅参与机体的过敏反应、自身免疫性疾病的发病机制，在肿瘤的发生、发展中也发挥一定作用（1-4）。
3、试剂盒组分：组分规格配制Cytokine standard 2瓶干粉状，按瓶上说明操作Biotinylated antibody 2管1：100用Dilution buffer R (1×)稀释Streptavidin-HRP 2管1：100用Dilution buffer R (1×)稀释Dilution buffer R (1×) 3瓶即用型Washing buffer (50×) 1瓶150∶用蒸馏水稀释TMB 1瓶即用型Stop solution 1瓶即用型Precoated ELISA plate 8×12 即用型封板膜2张即用型说明书1份4、需要实验者自行准备的试剂与仪器：1．酶标仪（建议参考仪器使用说明提前预热）2．微量加液器及吸头：P10，P50，P100，P200，P1000 3．蒸馏水或去离子水4．全新滤纸5．旋涡振荡器和磁力搅拌器5、注意事项：1.试剂应按标签说明储存，使用前室温平衡20-30分钟。
Xofigo 放射性治疗药剂指南说明书
UnitedHealthcare ® Medicare AdvantagePolicy GuidelineXofigo ® Radioactive Therapeutic AgentGuideline Number : MPG356.09Approval Date : November 8, 2023 Terms and ConditionsTable of Contents Page Policy Summary ............................................................................. 1 Applicable Codes .......................................................................... 1 References ..................................................................................... 2 Guideline History/Revision Information ....................................... 2 Purpose .......................................................................................... 3 Terms and Conditions . (3)See PurposeOverviewXofigo ® (radium Ra 223 dichloride) injection is an alpha particle-emitting radioactive therapeutic agent which mimics calcium and forms complexes with hydroxyapatite at areas of increased bone turnover, such as bone metastases.GuidelinesThe U.S. Food and Drug Administration (FDA) approved radium Ra 223 dichloride (Xofigo ® Injection, Bayer HealthCare Pharmaceuticals Inc.) for the treatment of patients with castration-resistant prostate cancer (CRPC), symptomatic bonemetastases and no known visceral metastatic disease.The recommended dose and schedule for Xofigo ® is 55 kBq/kg (1.49 microcuries/kg) administered by slow intravenous injection over 1 minute every 4 weeks for 6 doses.The following list(s) of procedure and/or diagnosis codes is provided for reference purposes only and may not be all inclusive. Listing of a code in this guideline does not imply that the service described by the code is a covered or non-covered health service. Benefit coverage for health services is determined by the member specific benefit plan document and applicable laws that may require coverage for a specific service. The inclusion of a code does not imply any right to reimbursement or guarantee claim payment. Other Policies and Guidelines may apply.CPT Code Description 79101 Radiopharmaceutical therapy, by intravenous administrationCPT ® is a registered trademark of the American Medical AssociationHCPCS Code DescriptionA9606 Radium RA-223 dichloride, therapeutic, per microcurieDiagnosis Code DescriptionC61Malignant neoplasm of prostateRelated Medicare Advantage Reimbursement Policy • Add-on Codes Policy, ProfessionalDiagnosis CodeDescriptionAnd at least one of the following:C79.51 Secondary malignant neoplasm of boneC79.52 Secondary malignant neoplasm of bone marrowCMS Local Coverage Determinations (LCDs) and ArticlesLCDArticleContractor Medicare Part A Medicare Part BN/A A54559 Billing and Coding: Xofigo Billing Instructions PalmettoAL, GA, NC, SC,TN, VA, WV N/AA55052 Billing and Coding: Radiopharmaceutical Agents Retired 12/29/2022WPSAK, AL, AR, AZ, CA, CO, CT, DE, FL, GA, HI, IA, ID, IL, IN, KS, KY, LA, MA, MD, ME, MI, MO, MS, MT, NC, ND, NE, NH, NJ, NM, NV, OH, OK, OR, PA, RI, SC, SD, TN, TX, UT, VA, VT, WA, WI, WV, WYIA, IN, KS, MI, MO, NECMS Benefit Policy ManualChapter 15; § 50 Drugs and BiologicalsCMS Claims Processing ManualChapter 12; § 30.5 Payment for Codes for Chemotherapy Administration and Nonchemotherapy Injections and Infusions Chapter 14; § 10 General Ambulatory Surgical CenterChapter 17; § 90.2 Drugs, Biologicals, and RadiopharmaceuticalsOther(s)CGS Website (Submitting Claims for Xofigo/Radium 223)CMS HCPCS Codes for which ASP Reporting is in Units of Measure Other Than an NDC, Updated July 2023, CMS Website Xofigo Package Insert, Bayer Healthcare Pharmaceuticals WebsiteRevisions to this summary document do not in any way modify the requirement that services be provided and documented in accordance with the Medicare guidelines in effect on the date of service in question.Date Summary of Changes11/08/2023Policy Summary OverviewRemoved and relocated language pertaining to the U.S. Food and Drug Administration (FDA)approval of radium Ra 223 dichloride (Xofigo ® Injection, Bayer HealthCare Pharmaceuticals Inc.) usage (refer to the Guidelines section) GuidelinesRevised language to indicate:Date Summary of Changeso The U.S. Food and Drug Administration (FDA) approved radium Ra 223 dichloride (Xofigo®Injection, Bayer HealthCare Pharmaceuticals Inc.) for the treatment of patients with castration-resistant prostate cancer (CRPC), symptomatic bone metastases and no known visceralmetastatic diseaseo The recommended dose and schedule for Xofigo® is 55 kBq/kg (1.49 microcuries/kg)administered by slow intravenous injection over 1 minute every 4 weeks for 6 dosesSupporting InformationUpdated References section to reflect the most current informationArchived previous policy version MPG356.08The Medicare Advantage Policy Guideline documents are generally used to support UnitedHealthcare Medicare Advantage claims processing activities and facilitate providers’ submission of accurate claims for the specified services. The document can be used as a guide to help determine applicable:Medicare coding or billing requirements, and/orMedical necessity coverage guidelines; including documentation requirements.UnitedHealthcare follows Medicare guidelines such as NCDs, LCDs, LCAs, and other Medicare manuals for the purposes of determining coverage. It is expected providers retain or have access to appropriate documentation when requested to support coverage. Please utilize the links in the References section above to view the Medicare source materials used to develop this resource document. This document is not a replacement for the Medicare source materials that outline Medicare coverage requirements. Where there is a conflict between this document and Medicare source materials, the Medicare source materials will apply.The Medicare Advantage Policy Guidelines are applicable to UnitedHealthcare Medicare Advantage Plans offered by UnitedHealthcare and its affiliates.These Policy Guidelines are provided for informational purposes, and do not constitute medical advice. Treating physicians and healthcare providers are solely responsible for determining what care to provide to their patients. Members should always consult their physician before making any decisions about medical care.Benefit coverage for health services is determined by the member specific benefit plan document* and applicable laws that may require coverage for a specific service. The member specific benefit plan document identifies which services are covered, which are excluded, and which are subject to limitations. In the event of a conflict, the member specific benefit plan document supersedes the Medicare Advantage Policy Guidelines.Medicare Advantage Policy Guidelines are developed as needed, are regularly reviewed and updated, and are subject to change. They represent a portion of the resources used to support UnitedHealthcare coverage decision making. UnitedHealthcare may modify these Policy Guidelines at any time by publishing a new version of the policy on this website. Medicare source materials used to develop these guidelines include, but are not limited to, CMS National Coverage Determinations (NCDs), Local Coverage Determinations (LCDs), Medicare Benefit Policy Manual, Medicare Claims Processing Manual, Medicare Program Integrity Manual, Medicare Managed Care Manual, etc. The information presented in the Medicare Advantage Policy Guidelines is believed to be accurate and current as of the date of publication and is provided on an "AS IS" basis. Where there is a conflict between this document and Medicare source materials, the Medicare source materials will apply.You are responsible for submission of accurate claims. Medicare Advantage Policy Guidelines are intended to ensure that coverage decisions are made accurately based on the code or codes that correctly describe the health care services provided. UnitedHealthcare Medicare Advantage Policy Guidelines use Current Procedural Terminology (CPT®), Centers for Medicare andMedicaid Services (CMS), or other coding guidelines. References to CPT® or other sources are for definitional purposes only and do not imply any right to reimbursement or guarantee claims payment.Medicare Advantage Policy Guidelines are the property of UnitedHealthcare. Unauthorized copying, use, and distribution of this information are strictly prohibited.*For more information on a specific member's benefit coverage, please call the customer service number on the back of the member ID card or refer to the Administrative Guide.。
ap1通路相关基因
ap1通路相关基因AP1通路是一种重要的信号传导途径，与许多生物学过程密切相关。
本文将从多个方面介绍与AP1通路相关的基因及其功能。
AP1通路中的关键基因包括c-Jun、c-Fos、JunB和FosB等。
这些基因是转录因子，能够调控基因的转录过程。
它们通过形成二聚体结合到靶基因的AP1位点上，从而调控相关基因的表达。
这些基因在细胞增殖、分化、凋亡等生物学过程中发挥重要作用。
c-Jun是AP1通路中最早被发现的基因之一。
它参与调控细胞周期、细胞增殖和细胞凋亡等过程。
c-Jun在细胞内的水平受多种信号通路的调控，包括MAPK信号通路。
c-Jun的高表达与多种癌症的发生和发展密切相关。
研究发现，抑制c-Jun的表达可以抑制肿瘤细胞的生长和转移。
c-Fos是另一个重要的AP1通路基因。
c-Fos与c-Jun形成二聚体后，共同调控多个基因的转录过程。
c-Fos在胚胎发育、细胞增殖和分化等过程中发挥重要作用。
研究发现，c-Fos的过度表达与多种疾病的发生有关，如肿瘤、心血管疾病等。
因此，c-Fos被认为是潜在的治疗靶点。
JunB是AP1通路中的另一个重要成员。
它在细胞分化和凋亡等过程中发挥重要作用。
JunB的表达水平受多种信号通路的调控，包括Wnt信号通路和TGF-β信号通路。
研究发现，JunB的缺失会导致胚胎发育异常和细胞增殖异常。
因此，JunB也被认为是治疗某些疾病的潜在靶点。
FosB是AP1通路中的另一个家族成员，它与c-Fos有相似的功能。
FosB的表达受多种信号通路的调控，包括神经递质信号通路和荷尔蒙信号通路。
FosB参与调控神经递质的合成和释放，对中枢神经系统的功能具有重要影响。
研究发现，FosB的过度表达与多种神经系统疾病的发生有关，如抑郁症、药物成瘾等。
因此，FosB也被认为是治疗这些疾病的潜在靶点。
除了上述基因外，AP1通路还与许多其他基因相关。
这些基因包括调节AP1通路的激酶、磷酸酶和蛋白酶等。
常见分子实验限制性内切酶酶切位点大全
AatII识别位点Acc65I识别位点AccI识别位点AciI识别位点AclI识别位点AcuI识别位点AfeI识别位点AflII识别位点AflIII识别位点AgeI识别位点AhdI识别位点AleI识别位点AluI识别位点AlwI识别位点AlwNI识别位点ApaI识别位点ApaLI 识别位点ApeKI 识别位点ApoI 识别位点AscI 识别位点AseI 识别位点AsiSI 识别位点AvaI 识别位点AvaII 识别位点AvrII 识别位点BaeI 识别位点BamHI识别位点BanI 识别位点BanII 识别位点BbsI 识别位点BbvCI 识别位点BbvI 识别位点BccI识别位点BceAI识别位点BcgI识别位点BciVI识别位点BclI识别位点BfaI识别位点BfuAI识别位点BglI识别位点BglII识别位点BlpI识别位点Bme1580I识别位点BmgBI识别位点BmrI识别位点BmtI识别位点BpmI识别位点Bpu10I识别位点BpuEI识别位点BsaAI识别位点BsaBI识别位点BsaHI识别位点BsaI识别位点BsaJI识别位点BsaWI识别位点BsaXI识别位点BseRI识别位点BseYI识别位点BsgI识别位点BsiEI识别位点BsiHKAI识别位点BsiWI识别位点BslI识别位点BsmAI识别位点BsmBI 识别位点BsmFI 识别位点BsmI 识别位点BsoBI 识别位点Bsp1286I 识别位点BspCNI 识别位点BspDI 识别位点BspEI 识别位点BspHI 识别位点BspMI 识别位点BspQI 识别位点BsrBI 识别位点BsrDI 识别位点BsrFI 识别位点BsrGI 识别位点BsrI 识别位点BssHII 识别位点BssKI 识别位点BssSI 识别位点BstAPI 识别位点BstBI 识别位点BstEII 识别位点BstNI 识别位点BstUI 识别位点BstXI 识别位点BstYI 识别位点BstZ17I 识别位点Bsu36I 识别位点BtgI 识别位点BtgZI 识别位点BtsCI 识别位点BtsI 识别位点Cac8I识别位点ClaI识别位点CspCI识别位点CviAII识别位点CviKI-1识别位点CviQI识别位点DdeI识别位点DpnI识别位点DpnII识别位点DraI识别位点DraIII识别位点DrdI识别位点EaeI识别位点EaeI识别位点EagI识别位点EarI 识别位点EciI 识别位点EcoNI 识别位点 EcoO109I识别位点EcoP15I 识别位点EcoRI 识别位点EcoRV 识别位点FatI 识别位点FauI 识别位点Fnu4HI 识别位点FokI 识别位点FseI 识别位点FspI 识别位点HaeII 识别位点HaeIIIHgaI 识别位点HhaI识别位点HincII识别位点HindIII识别位点HinfI识别位点HinP1I识别位点HpaI识别位点HpaII识别位点HphI识别位点Hpy188I识别位点Hpy188III识别位点Hpy99I识别位点HpyAV识别位点HpyCH4III识别位点HpyCH4IVHpyCH4V识别位点 KasI识别位点KpnI识别位点MboI 识别位点MboII 识别位点MfeI 识别位点MluI 识别位点MlyI 识别位点MmeI 识别位点MnlI 识别位点MscI 识别位点MseI 识别位点MslI 识别位点MspA1I 识别位点MspI 识别位点MwoI 识别位点NaeI 识别位点NarI 识别位点NciI 识别位点NcoI识别位点NdeI识别位点NgoMIV识别位点NheI识别位点NlaIII识别位点NlaIV识别位点NmeAIII识别位点NotI识别位点NruI识别位点NsiI识别位点NspI识别位点PacI识别位点PaeR7I识别位点PciI识别位点PflFI识别位点PflMI识别位点PhoI识别位点PleI识别位点PmeI识别位点PmlI识别位点PpuMI识别位点PshAI识别位点PsiI识别位点PspGI识别位点PspOMI识别位点PspXI识别位点PstI识别位点PvuI识别位点PvuII识别位点RsaI识别位点RsrII识别位点SacII识别位点SalI识别位点SapI识别位点Sau3AI识别位点Sau96I识别位点SbfI识别位点ScaI识别位点SexAI识别位点SfaNI识别位点SfcI识别位点SfiI识别位点SfoI识别位点SgrAI识别位点SmaI识别位点SnaBI识别位点SpeI识别位点SphI识别位点SspI识别位点StuI识别位点StyD4I识别位点StyI识别位点TaqαI识别位点TfiI识别位点TliI识别位点TseI识别位点Tsp45I识别位点Tsp509I识别位点TspMI识别位点TspRI识别位点Tth111I识别位点XbaI识别位点XcmI识别位点XhoI识别位点XmaI识别位点XmnI识别位点ZraI识别位点与大家共享（非原创）。
艾美捷小鼠白介素 12(IL-12)酶联免疫吸附测定试剂盒使用说明书
（本试剂盒仅供体外研究使用，不用于临床诊断！）产品货号：E-EL-M3062产品规格：96T/48T/24T/96T*5Elabscience®小鼠白介素12(IL-12)酶联免疫吸附测定试剂盒使用说明书Mouse IL-12(Interleukin 12) ELISA Kit使用前请仔细阅读说明书。
如果有任何问题，请通过以下方式联系我们：销售部电话************，************技术部电话************具体保质期请见试剂盒外包装标签。
请在保质期内使用试剂盒。
联系时请提供产品批号(见试剂盒标签)，以便我们更高效地为您服务。
用途该试剂盒用于体外定量检测小鼠血清、血浆或其它相关生物液体中IL-12浓度。
检测原理本试剂盒采用双抗体夹心ELISA法。
用抗小鼠IL-12抗体包被于酶标板上，实验时样品（或标准品）中的小鼠IL-12会与包被抗体结合。
后依次加入生物素化的抗小鼠IL-12抗体和辣根过氧化物酶标记的亲和素，抗小鼠IL-12抗体与结合在包被抗体上的小鼠IL-12结合，生物素与亲和素特异性结合而形成免疫复合物，游离的成分被洗去。
加入显色底物(TMB)，TMB在辣根过氧化物酶的催化下呈现蓝色，加终止液后变成黄色。
用酶标仪在450 nm波长处测OD值，IL-12浓度与OD450值之间呈正比，通过绘制标准曲线计算出样品中IL-12的浓度。
试剂盒组成及保存未拆封的试剂盒可在2-8℃保存一周；如果一周以后才使用试剂盒，请拆开试剂盒并按照下表中的条件分别保存各组分。
试剂体积以实际发货版说明书为准。
相关试剂在分装时会比标签上试验所需自备物品1.酶标仪(450 nm波长滤光片)2.高精度移液器，EP管及一次性吸头：0.5-10μL, 2-20μL, 20-200μL, 200-1000μL3.37℃恒温箱，4.双蒸水或去离子水5.吸水纸6.加样槽样品收集方法(具体处理方法可参考官网：/List-detail-241.html) 1.血清：全血样品于室温放置1小时或2-8℃过夜后于2-8℃，1000×g离心20分钟，取上清即可检测。
彭亨猪TNF-alpha ELISA试验簿说明书
Porcine TNF-alpha ELISA KitCatalog Number ES24RB (96 tests)Rev. 6Product descriptionThe Porcine TNF-alpha ELISA Kit is a solid-phase sandwich Enzyme-Linked Immunosorbent Assay (ELISA) designed to detect and quantify the level of porcine TNF-alpha in cell culture supernatants, plasma, and serum.Contents and storageUpon receipt, store at 2-8°C for 6 months or -20°C for 1 year.Components Cat. No. ES24RB(96 tests)Porcine TNF-alpha Antibody Coated wells, 96-well plate 1 platePorcine TNF-alpha Biotin Conjugate 2 vialsPorcine TNF-alpha Standard, recombinant porcine TNF-alpha 2 vialsWash Buffer Concentrate (20X)25 mLAssay Diluent C30 mLAssay Diluent B (5X)15 mL Streptavidin-HRP (600X)0.2 mLTMB Substrate12 mLStop Solution8 mL Adhesive Plate Covers2Materials required but not suppliedDistilled or deionized waterMicrotiter plater reader with software capable of measuring at 450 nmPlate washer-automated or manual (manifold dispenser)Calibrated adjustable precision pipettes and glass or plastic tubes for diluting solutionsProcedural guidelinesReview the Procedural guidelines and Plate washing directions in the ELISA Technical Guide at for details prior to starting the procedure.Reagents are lot-specific. Do not mix or interchange different reagent lots from various kit lots.Prepare 1X Wash Buffer1.Allow Wash Buffer Concentrate (20X) to reach room temperature and mix to redissolve any precipitated salts.2.Dilute 20 mL of the Wash Buffer Concentrate into 380 mL of deionized or distilled water. Label as 1X Wash Buffer.3.Store the concentrate and 1X Wash Buffer in the refrigerator. Use the diluted buffer within one month.Prepare diluentAssay Diluent B should be diluted 5-fold with deionized or distilled water before use.Prepare biotin conjugate1.Briefly spin down the biotin conjugate before use.2.Add 100 µL of 1X Assay Diluent B into the vial to prepare a biotin conjugate concentrate.3.Pipette up and down to mix gently (the concentrate can be stored at 4°C for 5 days).4.The biotin conjugate concentrate should be diluted 80-fold with 1X Assay Diluent B and used in step 2 of ELISA procedure.Sample preparation guidelinesCollect samples in pyrogen/endotoxin-free tubes.Freeze samples after collection if samples will not be tested immediately. Avoid multiple freeze-thaw cycles of frozen samples.Thaw completely and mix well (do not vortex) prior to analysis.Avoid the use of hemolyzed or lipemic sera. If large amounts of particulate matter are present in the sample, centrifuge or filter sample prior to analysis.Pre-dilute samplesAssay Diluent C should be used for dilution of serum, plasma, and cell culture supernatant samples.Dilute serum and plasma 2-fold.Because conditions may vary, it is recommended that each investigator determine the optimal dilution to be used for each application.Dilute standardsNote: Use glass or plastic tubes for diluting standards.1.Briefly spin down a vial of lyophilized standard.2.Add 400 µL Assay Diluent C into vial to prepare a 40 ng/mL standard solution. Dissolve the powder thoroughly by a gentle mix.Add 200 µL TNF-alpha standard from the vial, into a tube with 600 µL Assay Diluent C to prepare a 10 ng/mL standard solution.Pipette 400 µL Assay Diluent C into each tube. Use the stock standard solution to produce a dilution series (shown below). Mix each tube thoroughly before the next transfer. Assay Diluent C serves as the zero standard (0 ng/mL).200200 200 200 200 200 200DiluentVolume600 µL400 µL400 µL400 µL400 µL400 µL400 µL400 µL40 ng/mLStd110 ng/mLStd23.333 ng/mLStd31.111 ng/mLStd40.37 ng/mLStd50.123 ng/mLStd60.041 ng/mLStd70.014 ng/mLBlank0 ng/mLPrepare 1X Streptavidin-HRP solutionNote: Prepapre the Streptavidin-HRP within 15 minutes of usage.1.Briefly spin the Streptavidin-HRP and pipette up and down to mix gently before use, as precipitates may form during storage.2.Dilute Streptavidin-HRP 600-fold with 1X Assay Diluent B.3.Do not store diluted solution for future use.Perform ELISA (Total assay time: 4 hours and 45 minutes)Allow all reagents to reach room temperature before use. Mix all liquid reagents prior to use.IMPORTANT! Perform a standard curve with each assay.Determine the number of 8-well strips required for the assay. Insert the strips in the frames for use. Re-bag any unused strips and frames, and store at 2 to 8°C for future use.1Bind antigen a.For the standard curve, add 100 µL of standards to the appropriate wells (see Dilute standards). Forsamples, add 100 µL of diluted samples (see Dilute samples) to the wells.b.Cover wells and incubate for 2.5 hours at room temperature or over night at 4°C with gentle shaking.c.Discard the solution and wash 4 times with 1X Wash Buffer. Wash by filling each well with WashBuffer (300 µL) using a multi-channel Pipette or autowasher. Complete removal of liquid at each stepis essential for good performance. After the last wash, remove any remaining Wash Buffer byaspirating or decanting. Invert the plate and blot it against clean paper towels.2Add biotin conjugate a.Add 100 µL of prepared biotin conjugate (see Prepare biotin conjugate) to each well.b.Incubate for 1 hour at room temperature with gentle shaking.c.Discard the solution. Repeat the wash as in step 3.3Add Streptavidin-HRP a.Add 100 µL of prepared Streptavidin-HRP solution (see Prepare Streptavidin-HRP solution) to eachwell.b.Incubate for 45 minutes at room temperature with gentle shaking.c.Discard the solution. Repeat the wash as in step 3.4Add TMB substrate a.Add 100 µL of TMB Substrate to each well. The substrate will begin to turn blue.b.Incubate for 30 minutes at room temperature in the dark with gentle shaking.5Add stop solution Add 50 µL of Stop Solution to each well. Tap the side of the plate gently to mix. The solution in thewell changes from blue to yellow.Read the plate and generate the standard curve1.Read the absorbance at 450 nm. Read the plate within 30minutes after adding the Stop Solution.e curve-fitting software to generate the standard curve. Afour parameter algorithm provides the best standard curve fit.Optimally, the background absorbance may be subtracted from all data points, including standards, unknowns and controls,prior to plotting.3.Read the concentrations for unknown samples and controlfrom the standard curve. Multiple value(s) obtained forsample(s) by the appropriate factor to correct for the sampledilution.Note: Dilute samples producing signals greater than that of thehigest standard in Standard Diluent Buffer and reanalyze.Multiply the concentration by the appropriate dilution factor.Performance characteristicsStandard curve (example)These standard curves are for demonstration only. A standardcurve must be run with each assay.Intra-assay precisionTo determine intra-assay precision, two standard curves and 3 samples for each standard curve are run. The standard curve concentration points as well as the samples are tested in duplicates on a single plate. Two different concentration values are obtained for each sample, using the two separate standard curves. The two concentration values for each sample is compared to each otherusing the CV% calculation. Intra-Assay CV%: <10%Inter-assay precision To evaluate inter-assay precision, the second standard curve is tested on a separate plate along with the second set of samples. Inter-Assay CV%: <12%RecoverySample TypeAverage % Recovery Range (%)Cell Culture Supernatants 112102-121Plasma 9770-126Serum132123-142SpecificityThe sandwich ELISA antibody pair detects Porcine TNF-alpha. Linearity of dilutionThe cell culture supernatants, plasma, and serum samples were spiked with recombinant porcine TNF-alpha, serially diluted in sample diluent and evaluated. Observed values were compared to expected values to calculate percent recovery and demonstrate the dilution linearity of the assay.Sample TypeAverage % Expected Range (%)1:2 Dilution1:4 Dilution1:2 Dilution1:4 DilutionCell CultureSupernatants897678-9767-85 Plasma957585-10267-86Serum1037792-11868-88SensitivityThe minimum detecable dose of porcine TNF-alpha is 20 pg/mL.This was determined by assaying replicates of zero and thestandard curve. The mean signal of zero + 2 standard deviationsread in dose from the standard curve is the LLD. This value is thesmallest dose that is not zero with 95% confidence.Limited product warrantyLife Technologies Corporation and/or its affiliate(s) warrant theirproducts as set forth in the Life Technologies' General Terms andConditions of Sale found on Life Technologies' website at/us/en/home/global/terms-andconditions.html.If you have any questions, please contactLife Technologies at /support.Product label explanation of symbols and warningsCatalogNumberBatchCodeTemperaturelimitationUsebyManufacturerConsultinstructions for useCaution, consultaccompanying documentsDISCLAIMERTO THE EXTENT ALLOWED BY LAW, LIFE TECHNOLOGIES AND/OR ITS AFFILIATE(S) WILL NOT BE LIABLE FOR SPECIAL, INCIDENTAL, INDIRECT, PUNITIVE, MULTIPLE, OR CONSEQUENTIAL DAMAGES IN CONNECTION WITH OR ARISING FROM THIS DOCUMENT, INCLUDING YOUR USE OF IT. Important Licensing Information: These products may be covered by one or more Limited Use Label Licenses. By use of these products, you accept the terms and conditions of all applicable Limited Use Label Licenses.Corporate entity: Life Technologies Corporation | Carlsbad, CA 92008 USA | Toll Free in USA 1 800 955 6288©2021 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and its subsidiaries unless otherwise specified.For support visit /support or contact ************************.12-Apr-21。
新型冠状病毒主蛋白酶抑制剂的理论设计与筛选
新型冠状病毒主蛋白酶抑制剂的理论设计与筛选新型冠状病毒主蛋白酶抑制剂的理论设计与筛选新型冠状病毒（SARS-CoV-2）自2020年底以来迅速传播，导致全球大流行的新冠病毒病（COVID-19）。
面对这一严峻挑战，科学家们急切希望发现有效的治疗药物。
其中，主蛋白酶（Mpro）是SARS-CoV-2病毒在侵入人体细胞并复制自身时的关键酶类。
因此，针对Mpro的抑制剂研发成为一种重要的策略之一。
本文将探索新型冠状病毒主蛋白酶抑制剂的理论设计与筛选方法。
首先，我们需要了解主蛋白酶的结构特征。
主蛋白酶是一个双功能蛋白酶，包含了33个保守氨基酸残基，构成了两个主要的功能区域，即主底物结合口袋（S1）和主瓣杆区域（S2）。
S1区域是底物特异性区域，而S2区域则通过底物蛋白酶切口的产物调节酶活性。
这一了解为理论设计和筛选提供了重要的基础。
基于该结构特征，我们可以运用分子对接模拟（molecular docking）和分子动力学模拟（molecular dynamics simulation）来预测抑制剂与主蛋白酶之间的相互作用。
分子对接模拟可以预测抑制剂与酶活性区域的结合模式、亲和力和抑制效果，而分子动力学模拟可以模拟抑制剂与主蛋白酶复合物的动态变化情况，从而评估抑制剂的稳定性和可靠性。
同时，我们还可以利用计算机辅助药物设计（computer-aided drug design）技术进行大规模筛选。
结合已知信息，如蛋白质结构、配体结构和酶与配体结合位点的模拟结果，我们可以利用虚拟筛选（virtual screening）技术，寻找具有潜在抑制活性的化合物。
虚拟筛选可以从数千到数百万个化合物中快速筛选出潜在抑制剂。
在理论设计和筛选之后，我们还需要进行实验验证。
可利用细胞实验和动物模型验证候选抑制剂的有效性和安全性。
这些实验的结果将为药物的进一步优化和临床应用提供支持。
总结起来，新型冠状病毒主蛋白酶抑制剂的理论设计与筛选是一项有挑战性的任务。
1种用于检测抑制基因功能的酵母株系的建立(英文)
Ｌ ｕ— ，Ｇ Ｏ Ｑ －ｉ ｇ Ｉ ｉ Ｈ ｅ Ｕ ｉ ａ ｑｎ
（ ｎ ｔｕｅｏ ｌ ｅｕ Ｅ ｏｏｙ Ｔ ｂ ｔ ｇｉｕｔｒｌ ｎ ｎｍａ Ｈｕｂ ｎ ｒ ｏ ｅｅ ｙ ｇ ｈ ，Ｔｂ ｔ ６ ０ ０，Ｃ ｉａ） Ｉ ｓ ｔｔ ｆＰａ ａ ｃｌｇ ， ｉｅ Ａ ｒ ｌ ａ ａ ｄ Ａ ｉ ｌ ｓ ａ ｄｙ Ｃ ｌ ｇ ，Ｎ ｉ ｃ ｉ ｉｅ ０ ０ ｉ ｔ ｃ ｕ ｌ ｎ ８ ｈｎ
１种 用 于检 测 抑 制 基 因功 能 的 酵 母 株 系 的建 立
李 慧娥 ，郭其 强
（ 藏农牧学院高原生态研究所， 藏 林芝 西 西 ８００ ６ ００）
摘
要
酵 母 被 广 泛 用 于分 子 生 物 学 中基 因功 能 的 检 测 。 为 扩 大 酵 母 株 系 Ｕ Ｃ １ Ｃ ４ ９在 抑 制 基 因活 性 检 测 方
Ａ ｂｓｒ ｃ Ｓ ｃ ａ ｏ ｃ ｓｃ ｒｖｓａ ａ ｅ ｎ ｗｉｅ ｙ ｕ ｅ ｓ ｔｏｓ ｉ ｅ ｔｎ ｅ ｕ ｃ ｉｎ． Ｉ ｒ ｅ ｏ ｕ ｉｉｅ ａ Ｓ ｃ ｔａ ｔ ａｃ ｈ ｒ ｍｙ ｅ ｅｅ ｉｉ ｅ ｈ ｓ ｂ ｅ ｄ ｌ ｓ ｄ ａ ｏ ｌ ｎ ｔ ｓｉ ｇ ｇ ｎｅ ｆ ｎ ｔｏ ｎ ｏ ｄ ｒｔ ｔｌｚ ａ — ｃ ａ ｏ ｃ ｓｃ ｒｖｓａ ｔａｎ ＵＣＣ４１ ｏ ｅｓｉ ｇ ｒ ｐ ｅ ｓ ｒａ ｔｖ ｔｅ ｈ ｒ ｍｙ ｅ ｅｅ ｉｉ ｅ ｓｒ ｉ ９ ｆｒｔ ｔｎ ｅ ｒ ｓ ｏ ｃｉｉｉｓ，ｗｅ ｅ ｉｅ ｒ ｄ ＵＣＣ４１ ｌｔｎ ｔ ＥＵ２ ｇ ｎ ｎｇｎ ｅ ｅ ９ ｂｙｄｅｅｉ ｇ ｉｓＬ ｅｅ ａ ｔｔｅ ｓ ｍｅ ｔｍｅ ｒ ｐ ａ ｉ ｇ ｔ ｅ Ｌ ｎｄ ａ ｈ ａ ｉ ｅ ｌ ｃｎ ｈ ＥＵ２ ｗｉｈ ｔ 豫 Ｐ１ ｔ ｈｅ ｍａ ｋ ｒｖａ ｈｏ ｌｇ ｕ ｅ ｏ ｉ ａｉ ｎ．Ｔｈ ｅ ｓｒ ｉ ｅ ｉ— ｒ ｅ ｉ ｍｏｏ ｏ ｓ ｒ ｃ ｍｂ ｎ ｔｏ ｅ ｎ ｗ ｔａｎ ｄ ｓｇ
Guide-it Recombinant Cas9 (3
Takara Bio USA, Inc. 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: *******************************United States/Canada Asia Pacific Europe Japan Page 1 of 9Takara Bio USA, Inc. Guide-it™ Recombinant Cas9 (3 µg/µl) User ManualCat. Nos. 632640, 632641 (021121)Table of ContentsI. Introduction (3)A. Summary (3)II. List of Components (3)III. Additional Materials Required (3)A. Electroporation Supplies (3)B. Mammalian Cell Culture Supplies (4)C. General Supplies (4)D. sgRNA Development and Production (4)E. Detection and Characterization of Gene Editing (4)IV. Protocol Overview (5)V. Electroporation Protocol for Neon Transfection System (5)A. Protocol: Preparation of Cells and Media (5)B. Protocol: Preparation of Cas9-sgRNA RNP Complex (6)C. Protocol: Electroporation (6)VI. Electroporation Protocol for 4D-Nucleofector System (7)A. Protocol: Preparation of Cells (7)B. Protocol: Preparation of Cas9-sgRNA RNP Complex (8)C. Protocol: Electroporation (8)VII. References (9)Table of FiguresFigure 1. Protocol overview for Guide-it Recombinant Cas9 (3 µg/µl). (5)I. IntroductionA. SummaryThe CRISPR/Cas9 system has emerged as a powerful tool for gene editing because of its high targetingspecificity, editing efficiency, and ease of use in virtually any organism. CRISPR/Cas9 technologyconsists of two key components that form a complex: Cas9 endonuclease and a single guide RNA(sgRNA) that directs Cas9 to cleave genomic DNA in a sequence-specific manner (Jinek et al. 2012).This RNA-programmable method exploits the error-prone nature of the non-homologous end joiningDNA repair pathway (NHEJ) to generate gene knockouts (via insertion/deletion). The method can also beused to generate knockins via the homology-directed repair (HDR) pathway.CRISPR/Cas9 system components have been delivered successfully into target cells through a variety ofapproaches, including vector-based expression systems, transfection of RNA, and more recently,introduction of Cas9-sgRNA ribonucleoprotein (RNP) complexes. Delivery of Cas9-sgRNA RNPsprovides a fast turnaround for gene-editing experiments while minimizing the likelihood of off-targeteffects compared to vector-based approaches (Sander and Joung 2014), and this approach has beenoptimized for various cell types using microinjection, electroporation, and lipid-mediated transfection(Liang et al. 2015).Guide-it Recombinant Cas9 (3 µg/µl) is a recombinant wild-type Streptococcus pyogenes Cas9 nucleaseexpressed with a C-terminal nuclear-localization signal (NLS)and purified from E. coli for use inCRISPR/Cas9-mediated gene editing experiments. The Cas9 protein solution has been verified to besterile and well-tolerated by mammalian cells when electroporated as a ribonucleoprotein complex (RNP)with a single guide RNA (sgRNA) for knockout experiments, or as an RNP with a donor repair templatefor knockin experiments.II. List of ComponentsGuide-it Recombinant Cas9 (3 µg/µl) (Cat. No. 632641)•100 µg Guide-it Recombinant Cas9 (3 µg/µl)Guide-it Recombinant Cas9 (3 µg/µl) (Cat. No. 632640)• 3 x 100 µg Guide-it Recombinant Cas9 (3 µg/µl)•Store Guide-it Recombinant Cas9 (3 µg/µl) at –70°C.•Avoid repeated freeze/thaw cycles. We recommend preparing aliquots upon initial thawing of Guide-it Recombinant Cas9 (3 µg/µl).III. Additional Materials RequiredThe following reagents/materials are required but not included.A. Electroporation SuppliesUse of this product requires an electroporator, electroporation chamber (typically cuvettes or tips), and anelectroporation buffer that is suitable for your target cells. Here we provide separate guidelines for theNeon Transfection System (Thermo Fisher Scientific, Cat. No. MPK5000) and the 4D-NucleofectorSystem (Lonza, Cat. No. AAF-1002B).B. Mammalian Cell Culture Supplies•Culture medium, supplies, and additives specific to your target cells•Cell culture plates•PBS without Ca2+ or Mg2+•Trypsin/EDTA or equivalent•Humidified incubator (set at 37º C, 5% CO2)C. General Supplies•Single-channel pipettes•Nuclease-free thin-wall PCR tubes or stripsD. sgRNA Development and ProductionCRISPR/Cas9 gene editing requires a custom sgRNA with a user-designed targeting sequence that ishomologous to the target gene or genomic region of interest. Selecting an appropriate DNA sequence at the target region is critical for maximizing the potential for efficient cleavage at the target site and forminimizing the likelihood of non-specific cleavage events. There are several freely available online tools that can be helpful for determining suitable sgRNA target sequences for a given organism and genomic target. For a list of these tools, please refer to:/US/Products/Genome_Editing/CRISPR_Cas9/Resources/Online_tools_for_gui de_RNA_design.NOTE: For many applications, it is advisable to design and test several variant sgRNAs against the same genomic target region.Candidate sgRNAs must ultimately be produced in sufficient quantity for the generation of functionalCas9-sgRNA RNPs. For development and production of user-designed sgRNAs, we recommend either of the following kits:•For constructing and purifying sgRNAs: Guide-it sgRNA In Vitro Transcription Kit (Takara Bio, Cat.No. 632635).•For constructing and purifying sgRNAs, and testing target cleavage efficiencies in vitro: Guide-it Complete sgRNA Screening System (Takara Bio, Cat. No. 632636).E. Detection and Characterization of Gene EditingThese items are recommended for determining the efficiency of gene editing and the nature of the edits:Cat. No. Product Size631443 Guide-it Mutation Detection Kit 100 rxns631448 Guide-it Mutation Detection Kit 25 rxns632611 Guide-it Genotype Confirmation Kit 100 rxns631444 Guide-it Indel Identification Kit 10 rxnsIV. Protocol OverviewPlease read each relevant protocol completely before starting. Successful results depend on understanding and performing the following steps correctly.Figure 1. Protocol overview for Guide-it Recombinant Cas9 (3 µg/µl).V. Electroporation Protocol for Neon Transfection SystemHere we provide protocols for performing knockout and knockin experiments in hiPS cells and CD34-positive stem cells using the Neon Transfection System. While these protocols may serve as a helpful starting point forelectroporation of other cell types as well, further optimization will be required. Please refer to the Neon Transfection System User Manual and manufacturer’s website for detailed operating instructions for the Neon Transfection System.A. Protocol: Preparation of Cells and MediaCultured target cells are harvested, washed, and resuspended in the appropriate buffer.1.Prepare a sufficient number of fresh cells for your experiment.NOTE: Each electroporation requires 1 x 105 cells. However, due to the potential variation of pipetteand tip volumes, we recommend preparing 1.5X the necessary volume of cell suspension (i.e., 1.5 x 105cells) for electroporation with a 10-µl Neon Tip to ensure that there is sufficient volume.2.For hiPS cells (adherent cells), continue to Step3. For CD34-positive stem cells (suspension cells),skip to Step 5.3.Aspirate the medium, wash the cell layer once with PBS (without Ca2+ and Mg2+), and dissociate thecells using TrypLE Select Enzyme (1X) (Thermo Fisher Scientific, Cat. No. 12563011).4.Harvest the cells in growth medium.5.Take an aliquot of the cell suspension and measure the cell density using your preferred method.6.Harvest the cells by centrifugation at 400g for 5 min in a 15-ml conical tube.7.Wash the cells once with PBS (without Ca2+ and Mg2+), and then resuspend hiPS cells in Buffer R andCD34-positive stem cells in Buffer T (included with Neon kits) at a concentration of 2 x 107 cells/ml(i.e., 1.5 x 105 cells in 7.5 µl).NOTE: Use Resuspension Buffer R for established adherent and suspension cells as well as primaryadherent cells, and use Resuspension Buffer T for primary blood-derived suspension cells.8.Keep the cell suspension on ice until use.B. Protocol: Preparation of Cas9-sgRNA RNP ComplexCas9 and sgRNA components are combined to form RNP complexes for electroporation.1.Thaw Guide-it Recombinant Cas9 (3 µg/µl) and sgRNA solutions at room temperature.NOTE: We recommend preparing aliquots upon initial thawing of Guide-it Recombinant Cas9(3 µg/µl) to avoid repeated freeze/thaw cycles.bine the following components in a 200-µl PCR tube to mix the Cas9 protein and sgRNA at a 5:1mass ratio. The molar ratio of Cas9 protein to sgRNA will be approximately 1:1 in this mixture, andthe total volume will be 7.5 µl. Be sure to use the same buffer that was used to resuspend the cells.NOTE: The reaction volume indicated below is 1.5X the required volume.Per reaction:0.45 μl* sgRNA (e.g., 1 µg/µl)0.75 μl Guide-it Recombinant Cas9 (3 µg/µl)6.3 μl* Resuspension Buffer R or T7.5 μl Total volume*The added volume of sgRNA will vary depending on sgRNA concentration, and the added volume ofResuspension Buffer should be adjusted such that the total reaction volume is 7.5 µl. The volumesindicated above are based on a sgRNA concentration of 1 µg/µl.NOTE: Make a master mix if you are performing multiple electroporations.NOTE: The optimal amount of RNP complex may vary for different cell types.NOTE: To maximize electroporation efficiency, the combined volume of the Cas9 and sgRNA solutionsshould be ≤20% of the total volume of the Cas9-sgRNA RNP complex reaction (e.g., for the 7.5-µlreaction specified above, the combined volume of the sgRNA and Cas9 solutions should be ≤1.5 µl).NOTE: If you plan to use donor DNA to induce HDR-mediated knockin, add the DNA after thesubsequent incubation step (Step 3). We recommend using ≤1 µg of DNA for knockin experiments.Adjust the volume of Resuspension Buffer R or T included in the reaction such that the final volumeupon addition of donor DNA is 7.5 µl.3.Mix the reaction well by gently pipetting up and down. Incubate using a thermal cycler preheated to37°C with the following program:37°C 5 min4°C hold4.OPTIONAL: Add donor DNA and keep on ice until use.C. Protocol: ElectroporationCas9-sgRNA RNPs are electroporated into target cells.1.Fill the Neon Tube with 3 ml of Buffer E (included with Neon kits) and insert the Neon Tube into theNeon Pipette Station.ing the touchscreen on the Neon system, set up the electroporation parameters as follows:Pulse voltage / Pulse width / Pulse number = 1100 v / 20 ms / 2 pulsesNOTE: We have used these parameters successfully for hiPS cells and CD34-positive stem cellsusing the Neon Transfection System. Optimization of electroporation parameters will be required fordifferent target cell types. Suggested parameters for different cell types are included in thesupplementary material for (Liang et al. 2015).3.Gently resuspend the cells by tapping, and transfer 7.5 µl of the cell suspension into the PCR tubecontaining the 7.5 µl of Cas9-sgRNA RNP complex solution.4.Mix well by gently pipetting up and down.5.Insert the Neon Pipette into the Neon Tip and confirm that the pipette and tip are tightly connected.ing the Neon Pipette, aspirate the mixture slowly into the Neon Tip.NOTE: Avoid any air bubbles in the tip. If you notice air bubbles, place the sample back into thePCR tube and aspirate again into the tip without any air bubbles.7.Insert the Neon Pipette into the Neon Tube placed in the Neon Pipette Station and run the program.8.Remove the pipette very carefully and transfer the cells into a cell culture plate with pre-warmedmedium.NOTE: Use an appropriate well plate for your target-cell type. We had success using 24 and 48-wellplates for CD34-positive stem cells and hiPS cells, respectively. hiPS cells typically require greaterconfluence than regular adherent cells.9.Shake the plate appropriately to disperse the cells and incubate at 37°C in a humidified incubator with5% CO2 until the next necessary procedure.VI. Electroporation Protocol for 4D-Nucleofector SystemHere we provide protocols for performing knockout and knockin experiments in Jurkat and CD34-positive stem cells using the 4D-Nucleofector System with 16-well Nucleocuvette Strips. While these protocols may serve as a helpful starting point for electroporation of other cell types as well, further optimization will be required. Please refer to the 4D-Nucleofector System User Manual and manufacturer’s website for more detailed information.A. Protocol: Preparation of CellsCultured target cells are harvested, washed, and resuspended in the appropriate solution.1.Prepare a sufficient number of fresh cells for your experiment.NOTE: Each electroporation requires 2 x 105 cells.2.Take an aliquot of the cell suspension and measure the cell density using your preferred method.3.Harvest the cells by centrifugation at 400g for 5 min in a 15-ml conical tube.4.Wash once with PBS (without Ca2+ and Mg2+), and then resuspend Jurkat cells in SE NucleofectorSolution (with supplement) and CD34-positive stem cells in P3 Nucleofector Solution (withsupplement) at a concentration of 1 x 107 cells/ml (i.e., 2 x 105 cells in 20 µl).NOTE: Please refer to the 4D-Nucleofector System User Manual and manufacturer’s website formore information about working with other cell types.NOTE: 20 µl of cell suspension will be needed per well of the Nucleocuvette Strip.5.Keep the cell suspension on ice until use.B. Protocol: Preparation of Cas9-sgRNA RNP ComplexCas9 and sgRNA components are combined to form RNP complexes for electroporation.1.Thaw Guide-it Recombinant Cas9 (3 µg/µl) and sgRNA solutions at room temperature.NOTE: We recommend preparing aliquots upon initial thawing of Guide-it Recombinant Cas9(3 µg/µl) to avoid repeated freeze/thaw cycles.bine the following components in a 200-µl PCR tube to mix the Cas9 protein and sgRNA at a 5:1mass ratio:Per reaction (e.g. 5 µl):2μl* sgRNA (e.g., 1 µg/µl)3.3 μl Guide-it Recombinant Cas9 (3 µg/µl)5.3 μl Total volume*The added volume of sgRNA will vary depending on sgRNA concentration. The volume indicated aboveis based on an sgRNA concentration of 1 µg/µl.NOTE: Make a master mix if you are performing multiple electroporations.NOTE: The RNP volume required for efficient transfection needs to be optimized for different celltypes. Usually ≤10 µl of the RNP solution will be tested for electroporation in each well of the 16-well Nucleocuvette Strip.3.Mix the reaction well by gently pipetting up and down. Incubate using a thermal cycler preheated to37°C with the following program:37°C 5 min4°C HoldC. Protocol: ElectroporationCas9-sgRNA RNPs are electroporated into target cells.bel wells of Nucleocuvette Strips to be used for electroporation.bine 20 µl of cell suspension with 5 µl Cas9-sgRNA RNP complex solution in each well of theNucleocuvette Strip, and mix well by gently pipetting up and down.NOTE: If you plan to use donor DNA to induce HDR-mediated knockin, add the donor DNA at thisstep.3.Select the program CL-120 for Jurkat cells or the program D0-100 for CD34-positive stem cells.4.Insert the Nucleocuvette Strip into the Nucleofector machine and run the program.5.Add 80 µl of pre-warmed medium to each cuvette and allow electroporated cells to recover for 12min post-transfection at room temperature.6.Gently collect the cells along with the media from each well and transfer to individual wells of a 48-well plate containing pre-warmed medium.7.Shake the plate appropriately to disperse the cells and incubate at 37°C in a humidified incubator with5% CO2 until the next necessary procedure.VII. References1.Jinek, M., Chylinsky, K., Fonfara, I., Hauer, M., Doudna, J.A., & Charpentier, E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science. 337(6096), 816–21 (2012).2.Liang, X. et al. Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection. J.Biotechnol.208, 44–53 (2015).3.Sander, J.D. & Joung, J.K. CRISPR-Cas9 systems for genomic editing, regulation and targeting. Nat.Biotechnol. 32, 347-55 (2014).Contact UsCustomer Service/Ordering Technical Supporttel: 800.662.2566 (toll-free) tel: 800.662.2566 (toll-free)fax: 800.424.1350 (toll-free) fax: 800.424.1350 (toll-free)web: web: e-mail: **********************e-mail: ********************Notice to PurchaserOur products are to be used for Research Use Only. They may not be used for any other purpose, including, but not limited to, use in humans, therapeutic or diagnostic use, or commercial use of any kind. Our products may not be transferred to third parties, resold, modified for resale, or used to manufacture commercial products or to provide a service to third parties without our prior written approval.Your use of this product is also subject to compliance with any applicable licensing requirements described on the product’s web page at . It is your responsibility to review, understand and adhere to any restrictions imposed by such statements© 2021 Takara Bio Inc. All Rights Reserved.All trademarks are the property of Takara Bio Inc. or its affiliate(s) in the U.S. and/or other countries or their respective owners. Certain trademarks may not be registered in all jurisdictions. Additional product, intellectual property, and restricted use information is available at .This document has been reviewed and approved by the Quality Department.。
AP宏观经济考试英语大纲复习
AP宏观经济考试英语⼤纲复习The questions contained in this AP ? Macroeconomics Practice Exam are written to the contentspecifications of AP Exams for this subject. Taking this practice exam should provide students with an idea of their general areas of strengths and weaknesses in preparing for the actual AP Exam. Because this AP Macroeconomics Practice Exam has never been administered as an operational AP Exam, statistical data are not available for calculating potential raw scores or conversions into AP grades.This AP Macroeconomics Practice Exam is provided by the College Board for AP Exam preparation. Teachers are permitted to download the materials and make copies to use with their students in a class-room setting only. To maintain the security of this exam, teachers should collect all materials after their administration and keep them in a secure location. Teachers may not redistribute the files electronically for any reason.2008 The College Board. All rights reserved. College Board, Advanced Placement Program, AP , AP Central, SAT, and the acorn logo are registered trademarks of the College Board. PSAT/NMSQT is a registered trade-mark of the College Board and National Merit Scholarship Corporation. All other products and services may be trademarks of their respective owners. Visit the College Board on the Web: /doc/39d572536bec0975f565e25b.html .Practice ExamAdvanced Placement ProgramAP ?MacroeconomicsContentsDirections for Administration (ii)Section I: Multiple-Choice Questions (1)Section II: Free-Response Questions (13)Student Answer Sheet for Multiple-Choice Section (16)Multiple-Choice Answer Key (17)Free-Response Scoring Guidelines (18)The College Board: Connecting Students to College SuccessThe College Board is a not-for-profit membership association whose mission is to connectstudents to college success and opportunity. Founded in 1900, the association iscomposed of more than 5,000 schools, colleges, universities, and other educationalorganizations. Each year, the College Board serves seven million students and theirparents, 23,000 high schools, and 3,500 colleges through major programs and services incollege admissions, guidance, assessment, financial aid, enrollment, and teaching andlearning. Among its best-known programs are the SAT?, the PSAT/NMSQT?, and theAdvanced Placement Program? (AP?). The College Board is committed to the principlesof excellence and equity, and that commitment is embodied in all of its programs,services, activities, and concerns.Visit the College Board on the Web: /doc/39d572536bec0975f565e25b.html .AP Central is the official online home for the AP Program: /doc/39d572536bec0975f565e25b.html .AP? MacroeconomicsDirections for AdministrationThe AP Macroeconomics Exam is 2 hours and 10 minutes in length and consists of a multiple-choice sectionand a free-response section.The multiple-choice section is 1 hour and 10 minutes, contains 60 questions, and accounts for two-thirds of the final grade.The free-response section is 60 minutes, contains 3 questions, and accounts for one-third of the final grade. Ten minutes of the Section II time are reserved for reading the questions and planning answers.A 10-minute break should be provided after Section I is completed.The actual AP Exam is administered in one session. Students will have the most realistic experience if a complete morning or afternoon is available to administer this practice exam. If a schedule does not permit one time period for the administration of the entire practice exam, it would be acceptable to administer Section I one day and Section II on a subsequent day.Total scores on the multiple-choice section are based only on the number of questions answered correctly. No points are deducted for incorrect answers and no points are awarded for unanswered questions.The use of calculators, or any other electronic devices, is not permitted during the exam.It is suggested that the practice exam be completed using a pencil for Section I and a pen with black or dark blue ink for Section II to simulate an actual administration.Teachers will need to provide paper for the students to write their free-response answers. Teachers should give the students directions indicating how they wish the responses to be labeled so that the teacher will be able to associate the student’s response with the question the student intended to answer.Remember that students are not allowed to remove any materials, including scratch work, from the testing site.Section IMultiple-Choice QuestionsThe inclusion of source material in this exam is not intended as an endorsement by the College Board or ETS of the content, ideas, or values expressed in them. The material printed here reflects various aspects of the course of study on which this exam is based and is therefore appropriate to use to measure the skills and knowledge of this course. MACROECONOMICSSection ITime—70 minutes60 QuestionsDirections: Each of the questions or incomplete statements below is followed by five suggested answers or completions. Select the one that is best in each case and place the letter of your choice in the corresponding box on the student answer sheet.1. If a certain combination of goods or services liesoutside the production possibilities curve of aneconomy, which of the following is true?(A) Effective trade barriers have reduced foreignimports into the economy.(B) New technology is being used in production.(C) Resources are not available to achieve thatcombination of goods or services.(D) Resources are not being used efficiently toachieve that combination of goods orservices.(E) Resources are being used at a more rapid ratethan they were in the past.2. Which of the following groups would most likelygain from unanticipated inflation?(A) Landlords who own apartments in citieswith rent controls(B) Individuals who have fixed retirementincomes(C) Individuals who earn high incomes(D) Individuals who have borrowed money atfixed interest rates(E) Banks that have loaned all excess reservesat a fixed interest rate3. With a constant money supply, if the demand formoney decreases, the equilibrium interest rate and quantity of money will change in which of the following ways?I nterest Rate QuantityofMoney(A) Increase Decrease(B) I ncrease Not change(C) Decrease Decrease(D) Decrease I ncrease(E) Decrease Not change4. According to the graph above, an increase inaggregate supply will most likely cause incomeand employment to change in which of thefollowing ways?I ncome Employment(A) Decrease Decrease(B) Decrease I ncrease(C) No change I ncrease(D) I ncrease Decrease(E) I ncrease ncrease5. If the exchange rate between the United Statesdollar ($) and the British pound (￡) changed from $2 per ￡1 to $3 per ￡1, and domestic prices in both countries stayed the same, then the UnitedStates dollar would(A) depreciate, making United States importsfrom Britain more expensive(B) depreciate, making United States importsfrom Britain cheaper(C) appreciate, making United States importsfrom Britain more expensive(D) appreciate, making United States importsfrom Britain cheaper(E) purchase 3 times more British goods thanbefore the change occurred6. If an economy is operating with significantunemployment, an increase in which of thefollowing will most likely cause employmentto increase and the interest rate to decrease?(A) Purchases of government bonds by thecentral bank(B) Transfer payments(C) Reserve requirements(D) Government expenditures(E) Investment in basic infrastructure7. An increase in which of the following is mostlikely to promote economic growth?(A) Consumption spending(B) Investment tax credits(C) The natural rate of unemployment(D) The trade deficit(E) Real interest rates8. An appropriate fiscal policy to combat a recession would be to increase which of the following?(A) I nterest rates(B) The money supply(C) Taxes(D) Government spending(E) The sales of government bonds9. The concept of opportunity cost would no longer be relevant if(A) poverty in an economy no longer existed(B) the supply of all resources were unlimited(C) resources were allocated efficiently(D) real wages were flexible(E) all current incomes were invested in technological research10. An appreciation of the United States dollar on the foreign exchange market could be caused by a decrease in which of the following?(A) United States interest rates(B) The United States consumer price index(C) Demand for the dollar by United States residents(D) Exports from the United States(E) The tariff on goods imported into the United States 11. Which of the following would indicate that economic growth has occurred?(A) The production possibilities curve shifts tothe left.(B) The long-run aggregate supply curve shiftsto the right.(C) The aggregate demand curve shifts to the right.(D) The Phillips curve becomes flatter.(E) Business cycles no longer exist.12. Which of the following is most likely to occurif the Federal Reserve engages in open marketoperations to reduce inflation?(A) A decrease in interest rates(B) A decrease in reserves in the banking system(C) A decrease in the government deficit(D) An increase in the money supply(E) An increase in exports13. Which Federal Reserve action can shift theaggregate demand curve to the left?(A) Lowering the federal funds rate(B) Lowering income taxes(C) Lowering reserve requirements(D) Raising the discount rate(E) Raising government spending on nationaldefense14. Crowding out refers to the decrease in(A) national output caused by higher taxes(B) domestic production caused by increasedimports(C) private investment due to increased bor-rowing by the government(D) employment caused by higher inflation(E) exports caused by an appreciating currencyof a country15. If the real interest rate in the United Statesincreases relative to that of the rest of the world, capital should flow (A) into the United States and the dollar willdepreciate(B) into the United States and the dollar willappreciate(C) out of the United States and the dollar willdepreciate(D) out of the United States and the dollar willappreciate(E) out of the United States and the value of thedollar will not change16. Which of the following policy choices representsa combination of fiscal and monetary policiesdesigned to bring the economy out of a recession?(A) Decreasing both taxes and the money supply(B) Increasing both taxes and the money supply(C) Increasing government spending anddecreasing the federal funds rate(D) Increasing both taxes and the discount rate(E) Engaging in deficit spending and governmentbond sales17. Which of the following will be counted as unemployed by the United States Bureau of Labor Statistics?(A) Persons who quit their previous jobs to stayat home to care for sick parents(B) Persons who were laid off from their previousjobs and have not applied for a job in twoyears(C) Persons who were fired from their previousjobs and are actively applying for work(D) Persons who have given up looking for jobsafter long searches(E) Persons who quit their previous jobs to starttheir own businesses18. Which of the following sequences of eventswould occur if the Federal Reserve implemented contractionary monetary policy?(A) Interest rates increase, investment andconsumption spending decrease, aggregatedemand decreases, and output and pricesdecrease.(B) Interest rates increase, investment andconsumption spending decrease, aggregatedemand increases, and output and pricesdecrease.(C) Interest rates increase, investment andconsumption spending increase, aggregatedemand decreases, and output and pricesdecrease.(D) Interest rates decrease, investment andconsumption spending decrease, aggregatedemand decreases, and output and pricesdecrease.(E) Interest rates decrease, investment andconsumption spending decrease, aggregatedemand decreases, and output and pricesincrease. 19. Suppose that autonomous consumption is $400 and that the marginal propensity to consume is0.8. If disposable income increases by $1,200, consumption spending will increase by(A) $1,600(B) $1,360(C) $1,200(D) $ 960(E) $ 40020. In an economy in which all prices, includingwages, are completely flexible, an increase inlabor productivity will result in which of thefollowing changes in output and real wages?Output RealWages(A) I ncrease I ncrease(B) I ncrease Decrease(C) Decrease No change(D) Decrease I ncrease(E) Decrease Decrease21. When the average price level increases by 10 per-cent in a given year, which of the following must increase by 10 percent for real output to remain constant?(A) Real national income(B) Nominal national income(C) The international value of the currency(D) Real interest rates(E) Nominal interest rates22. Which of the following will occur in a competi-tive market when the price of a good is less than the equilibrium price?(A) Price will decrease to eliminate the surplusand restore equilibrium.(B) Price will decrease to eliminate the shortageand restore equilibrium.(C) Price will increase to eliminate the surplusand restore equilibrium.(D) Price will increase to eliminate the shortageand restore equilibrium.(E) Price will remain constant, because supplywill increase to eliminate the shortage.23. A short-run Phillips curve shows an inverserelationship between(A) interest rates and borrowing(B) inflation and unemployment(C) income and consumption(D) prices and quantity demanded(E) inputs and outputs24. Which of the following can be expected to causean increase in gross domestic product in theshort run?(A) An increase in the tax rate(B) An increase in the interest rate(C) Equal increases in both imports and exports(D) Equal increases in both taxes and governmentexpenditures(E) Equal decreases in both investment andgovernment expenditures25. If the federal government reduces its budgetdeficit when the economy is close to full employ-ment, which of the following will most likely result?(A) I nflation will increase.(B) Tax revenues will increase.(C) Interest rates will decrease.(D) Unemployment will decrease.(E) The international value of the dollar willincrease.26. Which of the following will cause the UnitedStates dollar to depreciate relative to the euro?(A) An increase in household income in theUnited States(B) An increase in interest rates in the UnitedStates(C) An increase in household income in Europe(D) A decrease in interest rates in Europe(E) A decrease in price level in the United States27. Stagflation is most likely to be caused by(A) an increase in aggregate demand(B) a decrease in aggregate demand(C) an increase in aggregate supply(D) a decrease in aggregate supply(E) a large increase in the money supply 28. Assume that the nominal interest rate is10 percent. If the expected inflation rate is5 percent, the real interest rate is(A) 0.5%(B) 2%(C) 5%(D) 10%(E) 15%29. Which of the following will lead to an increase inthe United States gross domestic product?(A) More individuals prepare their own personalincome tax forms.(B) Some citizens begin working abroad ascomputer programmers.(C) The government prohibits the sale ofalcoholic beverages.(D) Foreign companies build new assemblyplants in the United States.(E) A million United States households sell theirused cars to their children.30. An advance in technology will cause the(A) aggregate demand curve to shift to the right(B) aggregate demand curve to shift to the left(C) short-run aggregate supply curve to shift tothe left(D) long-run aggregate supply curve to shift tothe left(E) long-run aggregate supply curve to shift tothe right31. Suppose that the Federal Reserve buys$400 billion worth of government securitiesfrom the public. If the required reserve ratio is20 percent, the maximum increase in the moneysupply is(A) $1,600 billion(B) $1,800 billion(C) $2,000 billion(D) $2,200 billion(E) $2,400 billionQuestions 32-34 are based on the diagram below, which shows the production alternatives of two countries, Alpha and Beta, producing two goods, grain and steel, using all of their available resources.32. Before specialization and trade, the domesticopportunity cost of producing 1 ton of grain inAlpha and in Beta is which of the following?Alpha Beta(A) 1 ton of steel 1 ton of steel(B) 1 ton of steel 2 tons of steel(C) 2 tons of steel 1 ton of steel(D) 1 ton of steel 0.5 ton of steel(E) 0.33 ton of steel 1.5 tons of steel 33. The theory of comparative advantage implies that Alpha would find it advantageous to(A) export grain and import steel(B) export steel and import grain(C) export both grain and steel and importnothing(D) import both grain and steel and exportnothing(E) trade 1 ton of grain for 0.5 ton of steel34. At what real exchange ratio, also referred to as theterms of trade, between grain (G) and steel (S)would both Alpha and Beta find it mutuallyadvantageous to specialize and trade?(A) 1G = 3.0S(B) 1G = 1.5S(C) 1G = 1.0S(D) 1G = 0.5S(E) There is no real exchange ratio that wouldenable both countries to benefit, sinceAlpha has an absolute advantage in bothgoods.35. According to the graph above, which of the following is true about the long-run equilibrium of the economy depicted?(A) The economy is in long-run equilibrium.(B) The aggregate demand curve will shift to the left torestore long-run equilibrium.(C) The long-run aggregate supply curve will shift to theright to restore long-run equilibrium.(D) Without a fiscal policy stimulus, the economy willremain in a recession.(E) As wages increase, the short-run aggregate supply curve will shift to the left to restore long-run equilibrium.36. An increase in personal income taxes will mostlikely cause aggregate demand and aggregatesupply to change in which of the following ways in the short run? AggregateDemand AggregateSupply(A) Not change Decrease(B) Not change I ncrease(C) Decrease Not change(D) Decrease I ncrease(E) I ncrease Not change37. Which type of unemployment would increase ifworkers lost their jobs because of a recession?(A) Cyclical(B) Frictional(C) Seasonal(D) Search(E) Structural38. Which of the following is true about the marginalpropensity to consume?(A) It is the percentage of total income that isspent on consumption.(B) It determines the size of the simple spendingmultiplier.(C) It increases as incomes increase becauseincreases in income cause people to spendmore.(D) It is the same as the money multiplier.(E) It is equal to the average propensity toconsume for people with low incomes. 39. When an economy is operating below the full-employment level of output, an appropriatemonetary policy would be to increase which ofthe following?(A) The discount rate(B) The required reserve ratio(C) The international value of the dollar(D) Open market purchases of government bonds(E) Government expenditure on goods andservices 40. Assume that the economy is at full employment.Policymakers wish to maintain the price level but want to encourage greater investment. Which of the following combinations of monetary and fiscal policies would best achieve this goal? MonetaryPolicy FiscalPolicy(A) No change Contractionary(B) Expansionary No change(C) Expansionary Contractionary(D) Expansionary Expansionary(E) Contractionary Expansionary41. In one year, spending on consumption, invest-ment, and government purchases was equalto 103 percent of a country’s gross domesticproduct. This would be possible only if(A) the money supply increased(B) net exports were positive(C) net exports were negative(D) the government ran a budget surplus(E) the government had a balanced budget42. When firms restructure their operations todecrease production costs, the aggregate supplycurve, the price level, and real output will change in which of the following ways?AggregateSupplyCurve PriceLevel Real Output(A) Shift to the left Increase Increase(B) Shift to the left Increase No change(C) Shift to the right Increase Increase(D) Shift to the right Decrease Increase(E) Shift to the right Decrease Decrease43. An economy is in a short-run equilibrium at alevel of output that is less than full-employmentoutput. If there were no fiscal or monetary policy interventions, which of the following changes in output and the price level would occur in the long run?Output PriceLevel(A) I ncrease Decrease(B) I ncrease I ncrease(C) Decrease Decrease(D) Decrease I ncrease(E) No change No change44. Assume that the world operates under a flexible exchange rate system. If the central bank ofMexico increases its money supply but other countries do not change theirs, Mexico’s inflation rate and the international value of the Mexicanpeso will most likely change in which of the following ways?I nternationalI nflationRate Value of the Peso(A) I ncrease Appreciate(B) I ncrease Depreciate(C) I ncrease No change(D) Decrease Appreciate(E) Decrease Depreciate45. The Federal Reserve decreases the federal funds rate by(A) decreasing the reserve requirement(B) decreasing the discount rate(C) increasing the discount rate(D) selling government bonds on the open market(E) buying government bonds on the open market Labor Market Data for Country X(in millions of persons)Population180 Employed94Unemployed6 Not in labor force 8046. Based on the information in the table above, what is the unemployment rate for Country X?(A) 3.3%(B) 4.0%(C) 6.0%(D) 6.38%(E) 7.5% 47. Suppose that the government decreases taxes and at the same time the central bank decreases thediscount rate. The combined actions will result in(A) an increase in unemployment and a decreasein the interest rate(B) an increase in unemployment and an increasein the interest rate(C) an increase in the real gross domestic productand a decrease in the interest rate(D) an increase in the real gross domestic productand an increase in the interest rate(E) an increase in the real gross domestic productand an indeterminate change in theinterest rate48. In a closed economy with only lump-sumtaxation, if the marginal propensity to con-sume is equal to 0.75, a $70 billion increasein government spending could cause a max-imum increase in output of(A) $52.5 billion(B) $70 billion(C) $122.5 billion(D) $210 billion(E) $280 billion49. Which of the following is NOT a function of fiatmoney?(A) A standard of deferred payment(B) A unit of account(C) A source of intrinsic value(D) A store of value(E) A medium of exchange50. When an economy is at full employment, whichof the following will most likely create demand-pull inflation in the short run?(A) An increase in the discount rate(B) An increase in personal income taxes(C) A decrease in the real rate of interest(D) A decrease in government spending(E) A decrease in the money supply51. Under rational expectations, an announced expan-sion in the money supply will change nominal and real gross domestic products (GDP) in which of the following ways?NominalGDP RealGDP(A) I ncrease I ncrease(B) I ncrease Decrease(C) I ncrease No change(D) No change Decrease(E) No change No change52. A decrease in labor productivity will shift the(A) aggregate demand curve to the right(B) aggregate demand curve to the left(C) long-run aggregate supply curve to the right(D) short-run aggregate supply curve to the right(E) short-run aggregate supply curve to the left53. In the long run, if aggregate demand decreases,real gross domestic product (GDP) and the pricelevel will change in which of the following ways?PriceRealGDP Level(A) Decrease Decrease(B) Decrease I ncrease(C) No change Decrease(D) I ncrease Decrease(E) No change ncrease54. Suppose that all banks keep only the minimumreserves required by law and that there are nocurrency drains. The legal reserve requirement is10 percent. If Maggie deposits the $100 bill shereceived as a graduation gift from her grandmother into her checking account, the maximum increase in the total money supply will be(A) $10(B) $100(C) $900(D) $1,000(E) $1,100 55. Assuming fixed exchange rates, if country Z’srate of inflation increases relative to its tradingpartners, Country Z’s imports and exports willmost likely change in which of the followingways?I mports Exports(A) Decrease Decrease(B) Decrease I ncrease(C) I ncrease Decrease(D) I ncrease I ncrease(E) No change No change56. Which of the following household purchases willbe counted as part of gross private investment in a country’s gross domestic product?(A) Government bonds(B) Shares of a company stock(C) Corporate bonds(D) A new car for personal use(E) A newly constructed home57. An increase in aggregate demand will causewhich of the following?(A) A movement along a given short-runPhillips curve(B) The long-run Phillips curve to becomehorizontal(C) The short-run Phillips curve to shift tothe left(D) The long-run Phillips curve to shift tothe right(E) The long-run Phillips curve to shift tothe left58. Which of the following would cause the short-runaggregate supply curve to shift to the right?(A) An increase in the wage rate(B) An increase in the interest rate(C) An increase in the natural rate ofunemployment(D) A decrease in the capital stock(E) A decrease in the expected price level59. A decrease in business taxes would lead to anincrease in national income by increasing whichof the following?(A) The money supply(B) Unemployment(C) Aggregate demand only(D) Aggregate supply only(E) Both aggregate demand and aggregate supply 60. In an open economy, an increase in government budget deficit tends to cause the internationalvalue of a country’s currency and its trade deficit to change in which of the following ways?ValueofCurrency TradeDeficit(A) Appreciate Become smaller(B) Appreciate Become larger(C) Depreciate Become smaller(D) Depreciate Become larger(E) Not change Not changeEND OF SECTION IIF YOU FINISH BEFORE TIME IS CALLED, YOU MAYCHECK YOUR WORK ON THIS SECTION.。
MIQE Guidelines
定量PCR发展及遇到问题
AMPLIFICATION
-
大量的科研工作使用定量PCR，每年产生大量的文章，但是
如何合理设计实验没有共识 如何描述实验结果没有共识 （gene表达差异的柱状图，etc） 投稿时提供什么实验信息没有共识 （自身说服力差、编审犯难、同行无法重复、出现大量错误 结果）
AMPLIFICATION
/pcr
Reverse Transcription 反转录
AMPLIFICATION
/pcr
Reverse Transcription
AMPLIFICATION
RNA
cDNA
Reality Ideal ?
Reproducible Data Not Reproducible
– Need for both technical replicates and Biological replicates. – Number of replicates will depend on level of differences that are being presented. – Lower expression genes tend to require more replicates to establish statistical validity of small differences.
不管target gene的丰度如何、起始的RNA输入量如何，反转录的效率都是 一致的 确保cDNA的差异能够真实反映 RNA i.e. 基因表达的差异。 不 影响表达差异的判定。/Leabharlann crAMPLIFICATION
实验五 质粒DNA酶切(质粒限制性内切酶消化酶切)
AccⅠ识别序列
AccⅠ
能切割 T CCGG AGA
不能切割 T ×CCGG 6mATC
注意此处的区别
dam甲基化酶识别序列
采用去甲基化酶的大肠杆菌菌株来制备质粒DNA，可防止DNA 的甲基化。
③ 酶的星活性 (star activity)
又称第二活力，是指改变了酶切反应条件后特异序列识别特性降低的 一种现象。由于识别特异性的降低，可能对原识别序列相似的序列也产生 切割反应。
第三步：看多克隆位点（MCS）。它具有多个限制酶的单一切点。便于外源 基因的插入。
第四步：再看外源DNA插入片段大小。质粒一般只能容纳小于10Kb的外源 DNA片段。 一般来说，外源DNA片段越长，越难插入，越不稳定，转化效率越低。
第五步：是否含有表达系统元件，即：启动子——核糖体结合位点——克隆 位点——转录终止信号。 这是用来区别克隆载体与表达载体的。克隆载体中加入一些与表达调控有 关的元件即成为表达载体。
Eco RV (10442)
35S prom oter UTR
多克隆位点 pUC18 MCS lacZ promoter
35S prom oter
Eco RV (8842)
绿色荧光蛋白基因 GFP NOS 终止子加尾信号
T-DNA right border
HPT 潮霉素抗性基因
加尾信号，终止位UT点R T-DNA left border
10549 bp
Kan
Eco RV (6218)
pBNA中由于有较多的EcoR Ⅴ识别位点，因此，经 EcoR Ⅴ酶切后产生大小不一的条带，电泳后整个泳道呈现均一 的亮带。
酶切前的DNA
酶切后的DNA
实验材料和试剂
PARP抗体
PARP抗体产品编号产品名称包装AP102 PARP抗体 >20次产品简介：来源用途交叉反应性抗体识别位点PARP分子量Rabbit WB H, M, R Caspase剪切位点 116/89/24kD WB,blot.WesternH, human; M, mouse; R, rat.本PARP抗体(PARP antibody)为进口分装，用人工合成的PARP中一段包含Caspase剪切位点的多肽进行适当修饰后免疫rabbit，然后用protein A和抗原多肽亲和柱经过两步纯化而得到的高纯度抗体。
本PARP抗体可以识别全长的116kD PARP，也可以识别经Caspase剪切产生的89kD和24kD PARP片断。
未发现和其它相关蛋白有交叉反应。
PARP，即poly(ADP-ribose) polymerase，是定位在细胞核内，和应激条件下DNA修复密切相关的一种酶。
PARP在体外可以被多种Caspase剪切，在体内是Caspase 3的主要剪切对象。
对于人PARP，在Asp124和Gly215之间被Caspase剪切后，使PARP羧基端的催化结构域(89kD)和氨基端的DNA结合结构域(24kD)相分离，从而使PARP失去其酶活力。
PARP对于细胞的稳定和存活非常重要，PARP失去酶活力会加速细胞的不稳定。
PARP剪切被认为是细胞凋亡的一个重要指标，也通常被认为是Caspase 3激活的指标。
配套提供了Western一抗稀释液，可以用于Western检测时的一抗稀释。
建议本抗体用于Wetern检测时的起始稀释比例为1:1000(实际使用时需根据抗原水平的高低作适当调整)。
本抗体如果用于常规的Western检测至少可以检测20次。
包装清单：产品编号产品名称包装AP102-1 PARP抗体 20μlAP102-2Western一抗稀释液20ml—说明书1份保存条件：PARP抗体-20℃保存，Western一抗稀释液-20℃或4℃保存，一年有效。
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AP® Macroeconomics2012 Scoring GuidelinesThe College BoardThe College Board is a mission-driven not-for-profit organization that connects students to college success and opportunity. Founded in 1900, the College Board was created to expand access to higher education. Today, the membership association is made up of more than 5,900 of the world’s leading educational institutions and is dedicated to promoting excellence and equity in education. Each year, the College Board helps more than seven million students prepare for a successful transition to college through programs and services in college readiness and college success — including the SAT® and the Advanced Placement Program®. The organization also serves the education community through research and advocacy on behalf of students, educators, and schools. The College Board is committed to the principles of excellence and equity, and that commitment is embodied in all of its programs, services, activities, and concerns.© 2012 The College Board. College Board, Advanced Placement Program, AP, SAT and the acorn logo are registered trademarks of the College Board. All other products and services may be trademarks of their respective owners. Permission to use copyrighted College Board materials may be requested online at: /inquiry/cbpermit.html.Visit the College Board on the Web: .AP Central is the official online home for the AP Program: .Question 112 Points (2 + 6 + 4)(a)2 points:•One point is earned for a correctly labeled graph of the production possibilities curve (PPC).•One point is earned for showing point A inside the PPC.(b)6 points:•One point is earned for identifying the buying of bonds as the correct open-market operation to use.•One point is earned for drawing a correctly labeled graph of the money market.•One point is earned for showing a rightward shift of the money supply curve, resulting in a lower interest rate.•One point is earned for stating that the real interest rate will fall.•One point is earned for explaining that with the price level remaining constant, when the nominal interest rate falls, the real interest rate also falls.•One point is earned for stating that the real GDP will increase in the short run and explaining that investment or consumption increases, causing aggregate demand to increase.Question 1 (continued)(c)4 points:•One point is earned for stating that the current account deficit will increase.•One point is earned for explaining that the increase in real GDP increases income, which causes imports to increase and net exports to decrease.•One point is earned for stating that the international value of the bera will decrease.•One point is earned for explaining that the decline in the international value of the bera is due to an increase in the supply of the bera.Question 25 points (1 + 3 + 1)(a) 1 point:•One point is earned for calculating the correct reserve requirement of 10 percent ($10,000/$100,000).(b) 3 points:•One point is earned for stating that total bank reserves will decrease by $5,000.•One point is earned for stating that the $5,000 withdrawal has no effect on the M1 measure of the money supply because it only changes the composition of M1 between cash and demand deposits.•One point is earned for stating that the new value of the excess reserves is $500.(c) 1 point:•One point is earned for stating that the bank can borrow from the Federal Reserve or from another bank.Question 36 points (2 + 1 + 1 + 2)(a) 2 points:•One point is earned for drawing a correctly labeled graph and showing the AD and SRAS curves and PL1.•One point is earned for showing a vertical LRAS curve at the output Y1 through the intersection of the SRAS and AD curves.(b) 1 point:•One point is earned for showing a rightward shift of the aggregate demand curve and showing Y2 and PL2.(c) 1 point:•One point is earned for stating that real wages will fall because the price level has increased and the nominal wages are fixed in the short run.(d) 2 points:•One point is earned for stating that the investment component of AD will change.•One point is earned for stating that the long-run aggregate supply curve will shift to the right because the capital stock has increased.。