SMARTer PCR cDNA Synthesis Kit User Manual

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akara Bio Company

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SMARTer™ PCR

cDNA Synthesis Kit

User Manual

Cat. Nos. 634925 & 634926

PT4097-1

Published January 2012

United States/Canada

Asia Pacific

Europe

Japan

Clontech Laboratories, Inc.

A T

1290

Mountain View, CA 94043

Technical Support (US)

E-mail: tech@

SMARTer™ PCR cDNA Synthesis Kit User Manual

Table of Contents

I. List of Components ..................................................................................................................3

II. Additional Materials Required .................................................................................................4

III. Introduction & Protocol Overview ..........................................................................................5

IV. RNA Preparation & Handling ...................................................................................................7

A. General Precautions ............................................................................................................................7

B. 7

C. RNA Purity ........................................................................................................................................8

D. Assessing the Quality of the RNA Template ........................................................................................8

V. SMARTer cDNA Synthesis ........................................................................................................9

A. General Considerations .....................................................................................................................10

B. PRoToCol: First-Strand cDNA Synthesis ....................................................................................10

C. PRoToCol: cDNA Amplification by lD PCR .............................................................................12

VI. Analysis of cDNA Amplification Results ..............................................................................16

VII. Troubleshooting Guide ..........................................................................................................17

VIII. References ............................................................................................................................18

Appendix A: Protocols for PCR-Select™ ....................................................................................19

A. Additional Materials Required ..........................................................................................................19

B. PRoToCol: cDNA Amplification by lD PCR .............................................................................19

C. PRoToCol: Column Chromatography .........................................................................................22

D. PRoToCol: RsaI Digestion ..........................................................................................................23

E. PRoToCol: Purification of Digested cDNA ................................................................................23

F. Controls for PCR-Select cDNA Subtraction .....................................................................................25

G. Analysis of Results of SMARTer PCR cDNA Synthesis for PCR-Select cDNA Subtraction ..............25

H. Troubleshooting ................................................................................................................................27

Appendix B: Virtual Northern Blots ............................................................................................28

Appendix C: Protocol for Non-Directional Cloning of SMARTer cDNA ..................................29

A. Additional Materials Required ..........................................................................................................29

B. PRoToCol: ds cDNA Polishing ..................................................................................................29

List of Figures

Figure 1. Flowchart of SMARTer cDNA synthesis .....................................................................................5

Figure 2. Guide to using the SMARTer cDNA synthesis protocol for PCR-Select cDNA Subtraction,

Virtual Northerns, Non-Directional Cloning & library Construction, and other applications. .............9

Figure 3. optimizing PCR parameters for SMARTer cDNA synthesis. ...................................................15

Figure 4. Analysis for optimizing PCR parameters ...................................................................................16

Figure 5. optimizing PCR parameters for SMARTer cDNA synthesis for use with

Clontech PCR-Select ...........................................................................................................................21

Figure 6. Virtual Northern blot analysis of cDNA fragments expressed in cells producing γ-globin. ........28

List of Tables

Table I: Guidelines for Setting Up 12

Table II: Cycling Guidelines Based on Starting Material ..........................................................................13

Table III: Troubleshooting Guide for First-Strand cDNA Synthesis & SMARTer PCR Amplification .....17

Table IV: Troubleshooting Guide for Preparing SMARTer cDNA for Subtraction ...................................27

Protocol No. PT4097-1

Version No. 013012

Clontech Laboratories, Inc.

A Takara Bio Company

2