酵母双杂交手册(英文).pdf
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1、CLONTECHInnovat iveTools t oAccelerat eDiscoveryYeast Protocols HandbookPT3024-1(PR13103)Published 14 March 2001Cat alog#:K1612-1MATCHMAKER Two-Hybrid Syst em 3(many)MATCHMAKER GAL4 LibrariesK1615-1MATCHMAKER Library Const ruct ion&Screening KitK1603-1MATCHMAKER One-Hybrid Syst emNL4000AAMATCHMAKER
2、Random Pept ide Library5398-1GAL4 AD Monoclonal Ant ibody5399-1GAL4 DNA-BD Monoclonal Ant ibodyFOR RESEARCH USE ONLYYeast Protocols HandbookTable of ContentsI.Introduction 4II.Introduction to Yeast Promoters 5III.Culturing and Handling Yeast 10IV.Preparation of Yeast Protein Extracts 12A.General Inf
3、ormat ion 12B.Preparat ion of Yeast Cult ures for Prot ein Ext ract ion 12C.Preparat ion of Prot ein Ext ract s:Urea/SDS Met hod 13D.Preparat ion of Prot ein Ext ract s:TCA Met hod 15E.Troubleshoot ing 17V.Yeast Transformation Procedures 18A.General Informat ion 18B.Reag ent s and Mat erials Req uir
4、ed 19C.Tips for a Successful Transformat ion 20D.Int eg rat ing Plasmids int o t he Yeast Genome 20E.Small-scale LiAc Yeast Transformat ion Procedure 20F.Troubleshoot ing Yeast Transformat ion 22VI.a-and|3-Galactosidase Assays 23A.General Informat ion 23B.In vivo Plat e Assay Using X-g al in t he Me
5、dium 25C.Colony-lift Filt er Assay 25D.Liq uid Cult ure Assay Using ONPG as Subst rat e 26E.Liq uid Cult ure Assay Using CPRG as Subst rat e 27F.Liq uid Cult ure Assay Using a Chemiluminescent Subst rat e 28G.a-Gal Quant it at ive Assay 32VII.Working with Yeast Plasmids 34A.General Informat ion 34B.
6、Plasmid Isolat ion From Yeast 34C.Transforming E.co/wit h Yeast Plasmids 36VIII.Analysis of Yeast Plasmid Inserts by PCR 39A.General Informat ion 39B.Tips for Successful PCR of Yeast Plasmid Templat es 39IX.Additional Useful Protocols 42A.Yeast Colony Hybridizat ion 42B.Generat ing Yeast Plasmid Seg
7、 reg ant s 43C.Yeast Mat ing 44X.References 46XI.MATCHMAKER and Related Products 49APPENDICESA.Glossary of Technical Terms 50B.Yeast Genet ic Markers Used in t he MATCHMAKER Syst ems 52C.Media Recipes 53A.Yeast Media 53B.E.co li Media 56D.Solut ion Formulat ions 57E.Plasmid Informat ion 61F.Yeast Ho
8、st St rain Informat ion 64CLONTECH Laboratories,IProtocol#PT3024-1Version#PR13103Yeast Protocols HandbookTable of Contents continuedList of TablesTable I.Yeast Promot er Const ruct s Used t o Reg ulat e Report er Gene Expression in MATCHMAKER Plasmids and Host St rains6Table II.Yeast Promot er Const
9、 ruct s in t he MATCHMAKER Cloning Vect ors9Table III.Comparison of p-g alact osidase Assays25Table IV.Select ed Yeast Genes and Their Associat ed Phenot ypes52Table V.MATCHMAKER Report er Genes and Their Phenot ypes52Table VI.MATCHMAKER Two-Hybrid Syst em Cloning Vect ors61Table VILMATCHMAKER Two-H
10、ybrid Syst em Report er and Cont rol Plasmids62Table VIII.MATCHMAKER One-Hybrid Syst em Cloning,Report er&Cont rol Plasmids63Table IX.Yeast Report er St rains in t he MATCHMAKER One-and Two-Hybrid Syst ems64List of FiguresFig ure 1.Seq uence of GAL4 DNA-BD recog nit ion sit es in t he GAL1,GAL2,MEL1
11、 UASs and t he UASG 17_mer6Fig ure 2.Urea/SDS prot ein ext ract ion met hod14Fig ure 3.TCA prot ein ext ract ion met hod16Notice to PurchaserPract ice of t he t wo-hybrid syst em is covered by U.S.Pat ent s#5,283,173 and#5,468,614 assig ned t o t he Research Foundat ion of t he St at e Universit y o
12、f New York.Purchase of any CLONTECH t wo-hybrid reag ent does not imply or convey a license t o pract ice t he t wo-hybrid syst em covered by t hese pat ent s.Commercial ent it ies purchasing t hese reag ent s must obt ain a license from t he Research Foundat ion of t he St at e Universit y of New Y
13、ork before using t hem.CLONTECH is req uired by it s licensing ag reement t o submit a report of all purchasers of t wo-hybrid reag ent s t o SUNY St ony Brook.Please cont act Barbara A.Sawit sky of SUNY St ony Brook for license informat ion(Tel:516-632-4163;Fax:516-632-9839).All plasmids(except for
14、 pACT2,pAS2-1,pGADT7 and pGBKT7)are licensed from The Research Foundat ion of t he St at e Universit y of New York.pACT2,pAS2-1,and yeast st rains Y187 and Y190 are licensed from Baylor Universit y.pGBKT7 is a derivat ive of pODB-8 and is licensed from t he Universit e de Bordeaux.The pBridg e Three
15、-Hybrid Vect or is t he propert y of t he Inst it ut Nat ional de la Sant e et de la Recherche Medicale(INSERM).pGADT7 and CG-1945 are t he propert y of CLONTECH Laborat ories,Inc.AH109 is t he propert y of CLONTECH Laborat ories,Inc.,and is a derivat ive of PJ69-2A which is t he propert y of t he U
16、niversit y of Wisconsin Research Foundat ion(WARF).The Polymerase Chain React ion(PCR)process is covered by pat ent s owned by Hoffman-LaRoche and F.Hoffmann-La Roche,Lt d.This product is int ended t o be used for research purposes only.It is not t o be used for drug or diag nost ic purposes nor is
17、it int ended for human use.CLONTECH product s may not be resold,modified for resale,or used t o manufact ure commercial product s wit hout writ t en approval of CLONTECH.2001,CLONTECH Laborat ories,Inc.All rig ht s reserved.Protocol#PT3024-1Version#PRCLONTECH Laboratories,Inc.3Yeast Protocols Handbo
18、okI.IntroductionThe Yeast Prot ocols Handbook provides backg round informat ion and g eneral yeast prot ocols t hat complement our syst em-specific User Manuals.The prot ocols in t his Handbook have been opt imized wit h our yeast-based MATCHMAKER Two-Hybrid and One-Hybrid Syst ems,and MATCHMAKER Li
19、braries.The Yeast Prot ocols Handbook is especially useful for researchers who wish t o use yeast as a vehicle for t heir molecular biolog y experiment s,but have lit t le or no prior experience working wit h yeast.For novice and experienced users alike,t he Yeast Prot ocols Handbook will help you o
20、bt ain t he best possible result s wit h your MATCHMAKER and ot her yeast-relat ed product s from CLONTECH.This Handbook includes:det ailed informat ion on cult uring and handling yeast informat ion on t he yeast promot ers used in t he MATCHMAKER Syst ems t wo prot ocols for preparing prot ein ext
21、ract s from yeast q uant it at ive and q ualit at ive p-g alact osidase assays(for use wit h lacZyeast report er st rains)a simple,opt imized prot ocol for isolat ing plasmids from yeast PCR amplificat ion and yeast colony hybridizat ion prot ocols for t he rapid analysis of posit ive clones obt ain
22、ed in a library screening a small-scale,lit hium acet at e yeast t ransformat ion prot ocol addit ional prot ocols for working wit h cert ain yeast plasmids and host st rainsThe special applicat ion of yeast t ransformat ion for one-and t wo-hybrid library screening is covered in det ail in each pro
23、duct-specific User Manual.The special applicat ion of yeast mat ing for library screening is covered in t he Pret ransformed MATCHMAKER Libraries User Manual.About our yeast-based productsThe MATCHMAKER GAL4 Two-Hybrid Syst ems(#K1604-1,K1605-1,K1612-1)and LexA Two-Hybrid Syst em(#K1609-1)are comple
24、t e kit s for ident ifying and invest ig at ing prot ein-prot ein int eract ions in vivo using t he yeast t wo-hybrid assay.The MATCHMAKER One-Hybrid Syst em(#K1603-1)provides t he basic t ools for ident ifying novel prot eins in vivo t hat bind t o a t arg et DNA seq uence such as a c/s-act ing reg
25、 ulat ory element.MATCHMAKER Two-Hybrid Syst ems are compat ible wit h our pBridg e Three-Hybrid Vect or(#6184-1)for t he invest ig at ion of t ert iary prot ein complexes.The MATCHMAKER Libraries are const ruct ed in vect ors t hat express insert s as fusions t o a t ranscript ional act ivat ion do
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- 酵母 双杂交 手册 英文
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