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Manual: GAL4 Two-Hybrid Phagemid Vector Kits

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Manual: GAL4 Two-Hybrid Phagemid Vector Kits GAL4 Two-Hybrid Phagemid Vector Kits INSTRUCTION MANUAL Catalog #211351 (GAL4 Two-Hybrid Phagemid Vector Kit), #235700 (pAD-GAL4-2.1 Phagemid Vector Kit), and #235702 (pBD-GAL4 Cam Phagemid Vector Kit) Revision A For In Vitro Use Only 211351-12 LIMITED PRODUCT WARRANTY This warranty limits our liability to replacement of this product. No other warranties of any kind, express or implied, including without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided by Agilent. Agilent shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product. ORDERING INFORMATION AND TECHNICAL SERVICES United States and Canada Agilent Technologies Stratagene Products Division 11011 North Torrey Pines Road La Jolla, CA 92037 Telephone (858) 373-6300 Order Toll Free (800) 424-5444 Technical Services (800) 894-1304 Internet [email protected] World Wide Web www.stratagene.com Europe Location Telephone Fax Technical Services Austria 0800 292 499 0800 292 496 0800 292 498 Belgium 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 15775 0800 15740 0800 15720 France 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 919 288 0800 919 287 0800 919 289 Germany 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 182 8232 0800 182 8231 0800 182 8234 Netherlands 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 023 0446 +31 (0)20 312 5700 0800 023 0448 Switzerland 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 563 080 0800 563 082 0800 563 081 United Kingdom 00800 7000 7000 00800 7001 7001 00800 7400 7400 0800 917 3282 0800 917 3283 0800 917 3281 All Other Countries Please contact your local distributor. A complete list of distributors is available at www.stratagene.com. GAL4 Two-Hybrid Phagemid Vector Kits CONTENTS Materials Provided.............................................................................................................................. 1 Storage Conditions.............................................................................................................................. 1 Vectors................................................................................................................................... 2 Control Plasmids ................................................................................................................... 2 Host Strains ........................................................................................................................... 2 Additional Materials Required .......................................................................................................... 2 Reagents and Solutions..........................................................................................................2 Nucleic Acids ........................................................................................................................ 2 Equipment ............................................................................................................................. 3 Notice to Purchaser............................................................................................................................. 3 Introduction......................................................................................................................................... 4 Vectors.................................................................................................................................................. 6 The pAD-GAL4-2.1 Vector .................................................................................................. 7 The pBD-GAL4 Cam Vector ................................................................................................ 8 Host Strains ........................................................................................................................... 9 Preparation of Host Strains.............................................................................................................. 10 Bacterial Host Strain............................................................................................................ 10 Yeast Host Strain................................................................................................................. 11 Control Plasmids ............................................................................................................................... 13 Description .......................................................................................................................... 13 Applications......................................................................................................................... 13 Expected Results ................................................................................................................. 16 Activation Domain and DNA-Binding Domain Vector Construction .......................................... 17 Background ......................................................................................................................... 17 Target and Bait Protein Insert Preparation, Ligation, and Transformation ......................... 17 Yeast Transformation and Assay for Expression of Reporter Genes .................................. 19 Yeast Transformation....................................................................................................................... 20 Yeast Transformation Protocol............................................................................................ 21 Screening............................................................................................................................................ 25 Filter Lift Assay................................................................................................................... 25 Isolation of Plasmid DNA from Yeast .............................................................................................27 Appendix: General Comparison of Escherichia coli versus Yeast Host Strains ......................... 28 Troubleshooting ................................................................................................................................ 29 Two-Hybrid Vector System Screening................................................................................ 29 Plasmid Isolation from Yeast .............................................................................................. 30 Preparation of Media and Reagents................................................................................................ 31 Standard Media and Reagents ............................................................................................. 31 Two-Hybrid Vector System Media and Reagents............................................................... 32 References.......................................................................................................................................... 37 Endnotes............................................................................................................................................. 37 MSDS Information............................................................................................................................ 37 Quick-Reference Protocol ................................................................................................................ 40 Note The complete sequences for the pAD-GAL4-2.1 phagemid vector and the pBD-GAL4 Cam phagemid vector are available for downloading to your computer. The pAD-GAL4-2.1 and pBD-GAL4 Cam phagemid vector sequences are available from the GenBank® database (Accession #AF033313 and #U46126, respectively). GAL4 Two-Hybrid Phagemid Vector Kits MATERIALS PROVIDED Product Catalog # Materials provided GAL4 Two-Hybrid Phagemid Vector Kit 211351 Vectors pAD-GAL4-2.1 phagemid vector pBD-GAL4 Cam phagemid vector Control plasmids pGAL4 control plasmid pBD-WT control plasmid pAD-WT control plasmid pBD-MUT control plasmid pAD-MUT control plasmid pLamin C control plasmid Bacterial host strain XL1-Blue MRF´ straina Yeast host strain YRG-2 strain pAD-GAL4-2.1 Phagemid Vector Kit 235700 Vector pAD-GAL4-2.1 phagemid vector Bacterial host strain XL1-Blue MRF´ strain Yeast host strain YRG-2 strain pBD-GAL4 Cam Phagemid Vector Kit 235702 Vector pBD-GAL4 Cam phagemid vector Bacterial host strain XL1-Blue MRF´ strain Yeast host strain YRG-2 strain a Also available separately. STORAGE CONDITIONS Vectors: –20°C Control Plasmids: –20°C Bacterial Glycerol Stock: –80°C Yeast Glycerol Stock: –80°C Revision A © Agilent Technologies, Inc. 2008. GAL4 Two-Hybrid Phagemid Vector Kits 1 Vectors ♦ The pAD-GAL4-2.1 phagemid vector undigested (20 μg) ♦ The pBD-GAL4 Cam phagemid vector undigested (20 μg) Store the pAD-GAL4-2.1 and pBD-GAL4 Cam phagemid vectors at –20°C. Control Plasmids ♦ The pGAL4, pBD-WT, pAD-WT, pBD-MUT, pAD-MUT, and pLamin C control plasmids for the detection of protein–protein interactions in yeast [20 μg each at a concentration of 1 μg/μl in TE buffer (see Preparation of Media and Reagents)]. Store at –20°C. Host Strains For host strain storage conditions, see Preparation of Host Strains. Bacterial Host Strain XL1-Blue MRF´ Strain Δ(mcrA)183 Δ(mcrCB-hsdSMR-mrr)173 endA1 supE44 thi-1 recA1 gyrA96 relA1 lac [F´ proAB lacIqZΔM15 Tn10 (Tetr)] Yeast Host Strain YRG-2 Strain Mata ura3-52 his3-200 ade2-101 lys2-801 trp1-901 leu2-3 112 gal4-542 gal80-538 LYS2::UASGAL1-TATA
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