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1、Clontech Laboratories, Inc. A Takara Bio Company 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: United States/Canada 800.662.2566 Asia Pacific +1.650.919.7300 Europe +33.(0)1.3904.6880 Japan +81.(0)77.543.6116 Page 1 of 30 Clontech Laboratories, Inc. SMARTer RACE 5/3
2、Kit User Manual Cat. No(s). 634858, 634859 (022714) SMARTer RACE 5/3 Kit User Manual (022714) Clontech Laboratories, Inc. A Takara Bio Company Page 2 of 30 Table of Contents I. Introduction . 4 II. List of Components . 7 III. Additional Materials Required 8 IV. Primer Design . 9 A. Primer Sequence
3、. 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
4、 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-
5、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 RA
6、CE 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 SMARTer RACE 5/3 Kit User Manual (022714) Clontech Laboratories, Inc. A Takara Bio Company Page 3 of 30 Table of Figures
7、Figure 1. Mechanism of SMARTer cDNA synthesis . 4 Figure 2. Overview of the SMARTer RACE procedure . 6 Figure 3. The relationship of gene-specific primers to the cDNA template. 9 Figure 4. 5- and 3-RACE sample results . 22 Figure 5. Detailed mechanism of the 5-RACE reactions. 27 Figure 6. Detailed m
8、echanism of the 3-RACE reactions. 28 Table of Tables Table 1. Additional 5-RACE Sequence Obtained with SMART Technology . 5 Table 2. Setting up 5- and 3-RACE PCR Reactions . 15 Table 3. Troubleshooting Guide: Other Specific Problems 25 SMARTer RACE 5/3 Kit User Manual (022714) Clontech Laboratories
9、, Inc. A Takara Bio Company Page 4 of 30 I. Introduction The SMARTer RACE 5/3 Kit provides a method for performing both 5- and 3-rapid amplification of cDNA ends (RACE). The SMARTer RACE 5/3 Kit includes our SMARTer II A Oligonucleotide and SMARTScribe Reverse Transcriptase, which provides better se
10、nsitivity, less background and higher specificity than previous kits. This powerful system allows you to amplify the complete 5 sequence of your target transcript from as little as 10 ng of total RNA. The cornerstone of SMARTer RACE cDNA synthesis is SMART technology, which eliminates the need for p
11、roblematic adaptor ligation and lets you use first-strand cDNA directly in RACE PCR, a benefit that makes RACE far less complex and much faster (Chenchik et al., 1998). Additionally, the SMARTer RACE Kit exploits Clontechs technology for suppression PCR designed for compatibility with In-Fusion clon
12、ing) o 25 l Control 3-RACE TFR Primer (10 M; designed for compatibility with In-Fusion cloning) In-Fusion Cloning o 10 l Linearized pRACE (50 ng/l) General Reagents o 2 tubes Tricine-EDTA Buffer (1 ml each) SeqAmp DNA Polymerase (Cat. No. 638504) Store all components at 20C. 50 l SeqAmp DNA Polymera
13、se 1.25 ml SeqAmp PCR Buffer (2X) In-Fusion HD Cloning Kit (Cat. No. 639648) (Not sold separately) Store all components at 20C. 20 l 5X In-Fusion HD Enzyme Premix 5 l pUC19 Control Vector, linearized (50 ng/l) 10 l 2 kb Control Insert (40 ng/l) NucleoSpin Gel and PCR Clean-Up Kit (Cat. No. 740609.10
14、) (Not sold separately) Store all components at room temperature 10 ml Binding Buffer NTI 6 ml Wash Buffer NT3 (concentrate) 5 ml Elution Buffer NE (5 mM Tris/HCl, pH 8.5) 10 NucleoSpin Gel and PCR Clean-Up Columns (yellow rings) 10 Collection Tubes (2 ml) SMARTer RACE 5/3 Kit User Manual (022714)
15、Clontech Laboratories, Inc. A Takara Bio Company Page 8 of 30 Stellar Competent Cells (Cat. No. 636763) Store Stellar Competent Cells at 70C. Store all other components at 20C. 10 tubes Stellar Competent Cells (100 l/tube) 10 tubes SOC Medium (1 ml/tube) 10 l pUC19 Vector (0.1 ng/l) III. Additional
16、Materials Required If your RNA template is from a non-eukaryotic organism and lacks a polyadenylated tail, you can add one prior to first-strand 3-cDNA synthesis using the following enzyme: Poly(A) Polymerase (Takara Bio Cat. No. 2180A) The following materials are required for In-Fusion cloning and transformation, but not
