clontech试剂盒 SMARTer_RACE 5'/3' Kit User Manual
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IV. Introduction ..................................................................................................................................................................... 4 Additional Materials Required .................................................................................................................................... 8 Primer Design ............................................................................................................................................................. 9 II. List of Components ......................................................................................................................................................... 7
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 PCR ............................................................................................................................ 21
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 Priming ................................................................................ 29