SMARTer_RACE_5_3__Kit_User_Manual_022714(7)

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

II. List of Components

This section lists the components for Cat. No. 634858, a 10 reaction kit. The larger, 20 reaction kit (Cat. No.

634859) contains two of every item listed below.

SMARTer RACE 5’/3’ Kit Components (Cat. No. 634860) (Not sold separately)

Store SMARTer II A Oligonucleotide and Control Mouse Heart Total RNA at –70°C. Store all other components at –20°C.

First-Strand cDNA Synthesis

o 10 µl SMARTer II A Oligonucleotide (24 μM)

o 10 µl 3' RACE CDS Primer A (12 μM)

o 10 µl 5' RACE CDS Primer A (12 μM)

o 10 µl 10X Random Primer Mix (20 μM)

o 40 µl 5X First-Strand Buffer (RNAse-Free)

o 5 µl Dithiothreitol (DTT) (100 mM)

o 1 ml Deionized H2O

o 10 µl RNase Inhibitor (40 U/µl)

o 20 µl SMARTScribe Reverse Transcriptase (100 U/µl)

o 10 µl dNTP mix (20 mM)

5’- and 3’-RACE PCR

o 400 µl 10X Universal Primer A Mix (UPM)

o 50 µl Universal Primer Short (10 µM)

o 5 µl Control Mouse Heart Total RNA (1 µg/µl)

o 25 µl Control 5'-RACE TFR Primer (10 µM; designed for compatibility with In-Fusion cloning)

o 25 µl Control 3'-RACE TFR Primer (10 µM; designed for compatibility with In-Fusion cloning)

In-Fusion Cloning

o 10 µl Linearized pRACE (50 ng/µl)

General Reagents

o 2 tubes Tricine-EDTA Buffer (1 ml each)

SeqAmp DNA Polymerase (Cat. No. 638504)

Store all components at –20°C.

50 μl SeqAmp DNA Polymerase

1.25 ml SeqAmp PCR Buffer (2X)

In-Fusion HD Cloning Kit (Cat. No. 639648) (Not sold separately)

Store all components at –20°C.

20 μl 5X In-Fusion HD Enzyme Premix

5 μl pUC19 Control Vector, linearized (50 ng/μl)

10 μl 2 kb Control Insert (40 ng/μl)

NucleoSpin Gel and PCR Clean-Up Kit (Cat. No. 740609.10) (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)

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