Takara Bio USA In-Fusion® SMARTer® Directional cDNA Library Construction Kit User Manual Cat. No. 634933 (050421) Takara Bio USA, Inc. 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: [email protected] United States/Canada Asia Pacific Europe Japan Page 1 of 40 800.662.2566 +1.650.919.7300 +33.(0)1.3904.6880 +81.(0)77.565.6999 In-Fusion SMARTer Directional cDNA Library Construction Kit User Manual Table of Contents I. List of Components .................................................................................................................................................. 4 II. Additional Materials Required .................................................................................................................................. 5 III. Introduction .......................................................................................................................................................... 6 IV. RNA Preparation & Handling ..............................................................................................................................11 A. Assessing the Quality of the RNA Template ........................................................................................................11 V. SMARTer cDNA Synthesis by LD-PCR .................................................................................................................13 A. Recommended Products ......................................................................................................................................13 C. Protocol: First-Strand cDNA Synthesis from More than 50 ng of RNA ................................................................14 D. Protocol: First-Strand cDNA Synthesis from Less than 50 ng of RNA .................................................................15 E. Protocol: ds cDNA Synthesis by LD-PCR using Advantage 2 Polymerase ...........................................................16 VI. ds cDNA Purification and Size Fractionation .......................................................................................................19 A. Protocol: CHROMA SPIN + TE-1000 Column Chromatography .........................................................................20 VII. In-Fusion Cloning of SMARTer ds cDNA into pSMART2IFD ............................................................................22 A. Protocol: In-Fusion Cloning ................................................................................................................................22 VIII. Transformation of Recombinant Plasmids into E. coli ..........................................................................................23 A. Protocol: Transformation into Electrocompetent E. coli .......................................................................................23 B. Pooling Transformation Mixes for Your Library ..................................................................................................24 IX. Titrating Plasmid Libraries ..................................................................................................................................25 A. Library Titration Precautions ...............................................................................................................................25 B. Protocol: Plasmid Library Titration......................................................................................................................25 X. Amplification of Plasmid Libraries ..........................................................................................................................26 A. Agar Plate Preparation for Amplification .............................................................................................................26 B. Protocol: Library Amplification ...........................................................................................................................27 XI. Troubleshooting Guide ........................................................................................................................................28 XII. References...........................................................................................................................................................32 Appendix A: Typical Results of PCR and ds cDNA Synthesis .........................................................................................33 Appendix B: pSMART2IFD Linearized Vector Map and In-Fusion Cloning Site ............................................................34 Appendix C: Protocol for Library Screening by PCR.......................................................................................................35 Appendix D: Preparing Your Vector for In-Fusion SMARTer cDNA Library Cloning ....................................................36 A. Primer Design .....................................................................................................................................................36 B. Protocol: Preparation of a Linearized Vector by PCR ...........................................................................................36 Appendix E: SMARTer ds cDNA Synthesis by LD-PCR using Advantage HD ...............................................................39 (050421) takarabio.com Page 2 of 40 Takara Bio USA, Inc. In-Fusion SMARTer Directional cDNA Library Construction Kit User Manual Table of Figures Figure 1. The In-Fusion SMARTer Directional cDNA Library Construction Kit includes a complete protocol for cDNA synthesis, library construction, and library amplification. ................................................................................................. 7 Figure 2. Flow chart of the In-Fusion SMARTer Directional cDNA Library Construction Kit protocol. ............................ 8 Figure 3. The In-Fusion SMARTer Directional cDNA Library Construction Kit allows you to quickly and efficiently transfer your gene into multiple expression systems via In-Fusion cloning.......................................................................10 Figure 4. Optimizing PCR parameters for SMARTer cDNA synthesis. ............................................................................18 Figure 5. Typical results: ds cDNA PCR product before (Lane 1) & after (Lane 2) size fractionation by the CHROMA SPIN +TE-1000 Column. ................................................................................................................................................19 Figure 6. CHROMA SPIN column and collection tubes. .................................................................................................19 Figure 7. Typical results: ds cDNA synthesized using the SMARTer control reagents and LD PCR protocol. ..................33 Figure 8. pSMART2IFD linearized vector map and In-Fusion Cloning site. ....................................................................34 Figure 9. How to simultaneously linearize your vector and introduce In-Fusion SMARTer sequences. ............................37 Figure 10. Preparation of a linearized vector by PCR. ......................................................................................................38 Table of Tables Table I. Guidelines for Setting Up PCR Reactions ...........................................................................................................16 Table II. Cycling Guidelines Based on Starting Material .................................................................................................17 Table III. Guidelines for Setting Up In-Fusion Cloning Reactions ...................................................................................22 Table IV. Guidelines for Setting Up Transformation Mixes .............................................................................................23 Table V. Guidelines for Setting Up PCR Reactions .........................................................................................................39 (050421) takarabio.com Page 3 of 40 Takara Bio USA, Inc. In-Fusion SMARTer Directional cDNA Library Construction Kit User Manual I. List of Components Store CHROMA SPIN Columns and Deionized H2O at room temperature. Store Control RNA and SMARTer II A Oligonucleotide at –70°C. Store all other reagents at –20°C. In-Fusion SMARTer PCR cDNA Synthesis Kit (Cat No. 634934; not sold separately) • 10 µl SMARTer V Oligonucleotide (12 µM) 5'–AAGCAGTGGTATCAACGCAGAGTAXXXXX–3' Rsa I (X = undisclosed base in the proprietary SMARTer oligo sequence) • 10 µl 3’ In-Fusion SMARTer CDS Primer (12 μM) 5’–CGGGGTACGATGAGACACCATTTTTTTTTTTTTTTTTTTTVN–3’ (N = A, C, G, or T; V = A, G, or C) • 12 µl SMARTScribe™ Reverse Transcriptase (100 units/µl) • 40 µl 5X First-Strand Buffer (RNase-Free) 250 mM Tris (pH 8.3) 30 mM MgCl2 375 mM KCl • 50 µl DTT (dithiothreitol; 100 mM) • 100 µl dNTP mix (dATP, dCTP, dGTP, dTTP; 10 mM each) • 10 µl RNase Inhibitor (40 U/µl) • 5 µl Control Mouse Liver Total RNA (1.0 µg/µl) • 200 µl 5' PCR Primer II A (12 µM) • 200 µl 3’ In-Fusion SMARTer PCR Primer (12 μM) 5’– CGGGGTACGATGAGACACCA-3’ • 200 µl Sodium Acetate (3 M; pH 4.8) • 100 µl Glycogen (20 µg/µl) • 1 ml Deionized H2O (Milli-Q-filtered, not DEPC-treated) • 30 ml 1X Fractionation Column Buffer 10 mM Tris (pH 7.4) 30 mM NaCl 0.5 mM EDTA (pH 7.4) • 20 CHROMA SPIN™ +TE-1000 Columns In-Fusion Snap Assembly Master Mix (Cat. No. 638948) • 100 µl 5X In-Fusion Snap Assembly Master Mix • 25 µl pUC19 Control Vector, linearized (50 ng/µl)