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Manual: Hybrizap-2.1 XR Library Construction Kit and Hybrizap-2.1

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Manual: Hybrizap-2.1 XR Library Construction Kit and Hybrizap-2.1 HybriZAP-2.1 XR Library Construction Kit and HybriZAP-2.1 XR cDNA Synthesis Kit Instruction Manual Catalog #235612 (HybriZAP-2.1 XR Library Construction Kit) and #235614 (HybriZAP-2.1 XR cDNA Synthesis Kit) Revision C Research Use Only. Not for Use in Diagnostic Procedures. 235612-12 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 Email [email protected] World Wide Web www.genomics.agilent.com Telephone Location Telephone United States and Canada 800 227 9770 Austria 01 25125 6800 Benelux 02 404 92 22 Denmark 45 70 13 00 30 Finland 010 802 220 France 0810 446 446 Germany 0800 603 1000 Italy 800 012575 Netherlands 020 547 2600 Spain 901 11 68 90 Sweden 08 506 4 8960 Switzerland 0848 8035 60 UK/Ireland 0845 712 5292 All Other Countries Please visit www.genomics.agilent.com and click Contact Us HybriZAP-2.1 XR Library Construction Kit and HybriZAP-2.1 XR cDNA Synthesis Kit CONTENTS Materials Provided .............................................................................................................................. 1 Storage Conditions .............................................................................................................................. 3 Additional Materials Required .......................................................................................................... 3 Notice to Purchaser ............................................................................................................................. 4 Introduction ......................................................................................................................................... 5 Overview of HybriZAP-2.1 Two-Hybrid Library Screens .................................................... 6 Vectors .................................................................................................................................................. 8 HybriZAP-2.1 Vector Map .................................................................................................... 9 pAD-GAL4-2.1 Vector Map ............................................................................................... 10 pBD-GAL4 Cam Vector Map ............................................................................................. 11 Bacterial Host Strains ....................................................................................................................... 12 Bacterial Strain Genotypes .................................................................................................. 12 XL1-Blue MRF´ Bacterial Strain Description ..................................................................... 12 Recommended Media .......................................................................................................... 13 Establishing an Agar Plate Bacterial Stock ......................................................................... 13 Preparation of a –80°C Bacterial Glycerol Stock ................................................................ 14 Yeast Host Strain .............................................................................................................................. 15 Yeast Strain Genotype and Phenotypic Verification ........................................................... 15 Yeast Strain Description ...................................................................................................... 16 Preparation of the Yeast Host Strain ................................................................................... 17 Preparation of a –80°C Yeast Glycerol Stock ..................................................................... 17 Helper Phage ..................................................................................................................................... 18 Storing the Helper Phage ..................................................................................................... 18 Titering the Helper Phage .................................................................................................... 18 Amplifying the Helper Phage .............................................................................................. 19 Packaging Extracts ........................................................................................................................... 20 Control Plasmids ............................................................................................................................... 20 Description .......................................................................................................................... 20 Applications ......................................................................................................................... 21 Expected Results for Control Plasmid Assays .................................................................... 23 Activation Domain Vector Construction......................................................................................... 24 Background ......................................................................................................................... 24 cDNA Insert Preparation ..................................................................................................... 24 Ligation of cDNA into the HybriZAP-2.1 Vector ............................................................... 39 Packaging Reaction ............................................................................................................. 40 Verification of Insert Percentage and Size .......................................................................... 43 Amplification of the HybriZAP-2.1 Library ....................................................................... 43 In Vivo Excision of the pAD-GAL4-2.1 Phagemid Vector from the HybriZAP-2.1 Vector ...... 45 ExAssist Helper Phage and XLOLR Strain ......................................................................... 46 Mass Excision Protocol ....................................................................................................... 46 Amplification of the Excised Phagemid Library ................................................................. 49 Single-Clone Excision Protocol .......................................................................................... 50 DNA-Binding Domain Vector Construction ................................................................................... 52 Bait Protein Insert Preparation, Ligation, and Transformation ........................................... 52 Yeast Transformation and Assay for Expression of Reporter Genes .................................. 55 Yeast Transformation ....................................................................................................................... 56 Yeast Transformation Protocol ............................................................................................ 57 Screening ............................................................................................................................................ 62 Filter Lift Assay ................................................................................................................... 62 Verification of Interaction ................................................................................................................ 64 Isolation of Plasmid DNA from Yeast ................................................................................ 64 Verification of Specificity of Protein–Protein Interactions ................................................. 66 Appendix I: General Comparison of Escherichia coli versus Yeast Host Strains ...................... 67 Appendix II: RNA Purification and Quantification ...................................................................... 68 RNA Purification ................................................................................................................. 68 RNA Quantification............................................................................................................. 68 Appendix III: Methylmercury Hydroxide Treatment ................................................................... 69 Appendix IV: Alkaline Agarose Gels .............................................................................................. 70 The Slide Technique ............................................................................................................ 70 The Vertical Alkaline Agarose Technique .......................................................................... 71 Conventional Submerged Gels ............................................................................................ 71 Protocol ............................................................................................................................... 71 Appendix V: Ethidium Bromide Plate Assay—Quantitation of DNA ......................................... 72 Preparation of Ethidium Bromide Plates ............................................................................. 72 Preparation of Standards .....................................................................................................
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