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Nos. Many (102518)  \nTable of Contents  \nI. Introduction ..................................................................................................................................................................... 3  \nII. List of Components ......................................................................................................................................................... 5  \nIII. Additional Materials Required .................................................................................................................................... 5  \nIV. PCR and Experimental Preparation ............................................................................................................................ 6  \nA. Preparation of a Linearized Vector by Restriction Digestion ..................................................................................... 6  \nB. PCR Primer Design ..................................................................................................................................................... 6  \nC. PCR Amplification of Target Fragment...................................................................................................................... 8  \nD. Control Reactions........................................................................................................................................................ 9  \nV. Which Protocol Should You Follow? ............................................................................................................................. 9  \nVI. Protocol I: In-Fusion Cloning Procedure w/Spin-Column Purification...................................................................... 9  \nA. Procedure for Spin-Column Purification of PCR Fragments...................................................................................... 9  \nB. In-Fusion Cloning Procedure for Spin-Column Purified PCR Fragments................................................................ 10  \nVII. Protocol II: In-Fusion Cloning Procedure w/Cloning Enhancer Treatment ............................................................. 11  \nA. Procedure for Treating Unpurified PCR Fragments with Cloning Enhancer ........................................................... 11  \nB. In-Fusion Cloning Procedure for Cloning Enhancer-Treated PCR Fragments......................................................... 11  \nVIII. Transformation Procedure......................................................................................................................................... 12  \nProcedure for Transformation Using Stellar Competent Cells ......................................................................................... 12  \nIX. Expected Results ....................................................................................................................................................... 12  \nX. Troubleshooting Guide ................................................................................................................................................. 13  \nAppendix A. Quick In-Fusion Cloning Protocol .................................................................................................................. 14  \nIn-Fusion Cloning Procedure for a PCR-Amplified Vector & Fragment ......................................................................... 14  \nAppendix B. pUC19 Linearized Vector Information............................................................................................................ 15  \nTable of Figures  \nFigure 1. In-Fusion HD protocol overview............................................................................................................................. 4  \nFigure 2. Universal primer design for In-Fusion technology.................................................................................................. 7  \nF","cbCaiaBpczJB2cdO","https://ap.wps.com/l/cbCaiaBpczJB2cdO","pdf",788571,15,"English","# Introduction\n# Protocols and experimental preparation\n## PCR primer design and amplification\n## Protocol I: spin-column purification workflow\n## Protocol II: cloning enhancer treatment workflow\n# Transformation procedure\n# Expected results\n# Troubleshooting guide\n# Appendix A: Quick in-Fusion cloning protocol\n# Appendix B: pUC19 linearized vector information","[{\"question\":\"What is the core principle of In-Fusion HD cloning?\",\"answer\":\"In-Fusion HD cloning uses the In-Fusion enzyme to fuse DNA fragments precisely by recognizing engineered 15-bp overlaps at fragment ends.\"},{\"question\":\"How are 15-bp overlaps generated for cloning?\",\"answer\":\"15-bp overlaps are engineered by designing PCR primers that amplify the desired insert and create the overlap sequences needed for fusion.\"},{\"question\":\"Which protocols does the manual provide for different sample preparations?\",\"answer\":\"It provides Protocol I for PCR fragments with spin-column purification and Protocol II for unpurified PCR fragments treated with Cloning Enhancer, followed by respective In-Fusion cloning steps.\"}]","In-Fusion HD Cloning Kit User Manual | PDF",1792705594,38]