SMARTer_RACE_5_3__Kit_User_Manual_022714(2)

clontech试剂盒 SMARTer_RACE 5'/3' Kit User Manual

I.

III.

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

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