Clontech Laboratories, Inc.pupil怎么读
SMARTer® RACE 5’/3’
Kit Ur Manual
Cat. No(s). 634858, 634859
(022714)
Clontech Laboratories, Inc.
A Takara Bio Company
1290 Terra Bella Avenue, Mountain View, CA 94043, USA
U.S. Technical Support:
United Asia Pacific Europe Japan Page 1 of 30
I. Introduction (4)
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II. List of Components (7)
beyonce knowlesIII. 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. Asssing RNA Template Quality (12)
D. Protocol: First-Strand cDNA Synthesis (13)
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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)
Ap pendix 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)
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)
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Figure 4. 5'- and 3'-RACE sample results (22)
Figure 5. Detailed mechanism of the 5'-RACE reactions. (27)
Figure 6. Detailed mechanism of the 3'-RACE reactions. (28)
Table of Tables
Table 1. Additional 5'-RACE Sequence Obtained with SMART Technology (5)
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Table 2. Setting up 5'- and 3'-RACE PCR Reactions (15)
Table 3. Troubleshooting Guide: Other Specific Problems (25)
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™Rever Transcripta, which provides better nsitivity, less background and higher specificity than previous你最珍贵英文
kits. This powerful system allows you to amplify the complete 5’ quence 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 problematic adaptor ligation and lets you u fir
st-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 Clontech’s technology for suppression PCR & step-out PCR to increa the nsitivity and
reduce the background of the RACE reactions. You can u either poly A+ or total RNA as starting material for constructing full-length cDNAs, even of very rare transcripts.
The SMARTer RACE 5’/3’ Kit is an improved version of our original SMARTer RACE cDNA Amplification Kit, designed to accommodate larger RNA input volumes and perform more efficiently on challenging targets
(e.g., tho that are long, GC-rich, etc.). RACE PCR products are amplified with our highly robust SeqAmp™
DNA Polymera, and cloned into the linearized pRACE vector with In-Fusion® HD Cloning. The In-Fusion HD Cloning Kit, NucleoSpin Gel and PCR Clean-Up Kit, and Stellar™ Competent Cel ls are included for your
convenience in cloning RACE products.
SMART technology provides a mechanism for generating full-length cDNAs in rever transcription reactions (Zhu et al., 2001). This is made possible by the joint action of the SMARTer II A Oligonucleotide and
SMARTScribe Rever Transcripta. When the SMARTScribe RT reaches the 5’ end of the RNA, its terminal transfera activity adds a few additional nucleotides to the 3’ end of the first-strand cDNA (Figure 1).
Figure 1. Mechanism of SMARTer cDNA synthesis.First-strand cDNA synthesis is primed using a mo
dified oligo (dT) primer. After SMARTScribe Rever Transcripta (RT) reaches the end of the mRNA template, it adds veral nontemplated residues. The SMARTer II
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A Oligonucleotide anneals to the tail of the cDNA and rves as an extended template for SMARTScribe RT.
The SMARTer II A Oligonucleotide contains a terminal stretch of modified bas that anneal to the extended cDNA tail, allowing the oligo to rve as a template for the RT. SMARTScribe RT switches templates from the mRNA molecule to the SMARTer oligo, generating a complete cDNA copy of the original RNA with the additional SMARTer quence at the end. Since the template switching activity of the RT occurs only when the enzyme reaches the end of the RNA template, the SMARTer quence is typically only incorporated into full-length, first-strand cDNAs. This process guarantees that the u of high quality RNA will result in the formation of a t of cDNAs that have a maximum amount of 5’ quence (Table I).
Table 1. Additional 5'-RACE Sequence Obtained with SMART Technology
Human gene Size of mRNA
(kb)
Additional quence
(bp)*
Matches genomic
quences
Piccolo presynaptic cytomatrix protein 20.29 +59 yes
Dynein, cytoplasmic 1, heavy chain 1 14.36 +36 yes
Polycystic kidney dia 1 14.14 +21 yes
Solute carrier family 1 12.02 +73 yes Microtubule-associated protein 1A 10.54 +13 yes
Spectrin, beta, non-erythrocytic 10.24 +32 yes
Transferrin receptor 5.0 +25 yes
Interferon-α receptor 2.75 +17 yes
Smooth muscle g-actin 1.28 +31 yes
Following rever transcription, SMART technology allows first-strand cDNA to be ud directly in 5’- and
3’-RACE PCR reactions. Incorporation of universal primer binding sites in a single-step during first-strand cDNA synthesis eliminates the need for tedious cond-strand synthesis and adaptor ligation. This simple and highly efficient SMARTer cDNA synthesis method ensures higher specificity in amplifying your target cDNA. Suppression PCR & step-out PCR techniques are ud in combination with SMARTer technology to decrea background amplification in RACE PCR.
Requirements for SMARTer RACE cDNA Amplification
The only requirement for SMARTer RACE cDNA amplification is that you know at least 23–28 nucleotides (nt) of quence information in order to design gene-specific primers (GSPs) for the 5’- and 3’-RACE reactions. (Additional quence information will facilitate analysis of your RACE products.) This limited requirement makes SMARTer RACE ideal for characterizing genes identified through diver methods, including cDNA subtraction, differential display, RNA fingerprinting, ESTs, library screening, and more.
Us of SMARTer RACE cDNA Amplificationexeter
SMARTer RACE cDNA amplification is a flexible tool—many rearchers u this kit in place of conventional kits to amplify just the 5’ or 3’ end of a particular cDNA. Others perform both 5’- and 3’-RACE, and many then go on to clone full-length cDNAs using one of the two methods described in the latter part of this protocol. In many cas, rearchers obtain full-length cDNAs without ever constructing or screening a cDNA library.南昌翻译
