RACE说明书.pdf

Takara Bio USA, Inc. SMARTer® RACE 5’/3’ Kit User Manual Cat. Nos. 634858, 634859 (052617) Takara Bio USA, Inc. 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: techUS@ United States/Canada Asia Pacific Europe Japan Page 1 of 30 800.662.2566 +1.650.919.7300 +33.(0)1.3904.6880 +81.(0)77.565.6999 SMARTer® RACE 5’/3’ Kit User Manual Table of Contents I. Introduction 4 II. List of Components 7 III. Additional Materials Required 8 IV. Primer Design 9 A. Primer Sequence 9 B. Additional Considerations for Design 10 C. Location of Primer Sequences within Genes 10 D. Nested Primers 10 V. Generating RACE-Ready cDNA 11 A. General Considerations 11 B. Preparation and Handling of Total and Poly A+ RNA 11 C. Assessing RNA Template Quality 12 D. Protocol: First-Strand cDNA Synthesis 13 VI. Rapid Amplification of cDNA Ends (RACE) 15 A. Things You Should Know Before Starting RACE PCR Reactions 15 B. Protocol: Rapid Amplification of cDNA Ends (RACE) 15 VII. Characterization of RACE Products 17 A. Protocol: Gel Extraction with the NuceloSpin Gel and PCR Clean-Up Kit 17 B. Protocol: In-Fusion Cloning of RACE Products 18 C. Sequencing RACE Products 19 VIII. References 20 Appendix A. Troubleshooting Guide 21 A. Troubleshooting Touchdown PCR21 B. Multiple Band RACE Products 23 C. Other Specific Problems 25 Appendix B. Detailed Flow Chart of 5’ RACE 27 Appendix C. Detailed Flow Chart of 3’ RACE 28 Appendix D. 5’-RACE cDNA Amplification with Random Primers 29 A. Protocol: First-Strand cDNA Synthesis with Random Priming29 (052617)