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)