Features
Rapid amplification of 3’ cDNA ends
Contains a highly efficient blend of reverse transcriptase and BIO-X-ACT™ Long DNA Polymerase

Applications
Studying alternative splicing forms of mRNA
Identification of translation termination codon
Characterizing 3´ untranslated sequence
Obtaining the full sequence of a gene

Description

The 3' RACE Kit employs the rapid amplification of cDNA ends (RACE) PCR method for obtaining the full-length 3' cDNA sequence from a known partial cDNA sequence. Known sources of partial cDNA sequence include interaction trap assays (e.g. yeast 2 hybrid system), differential display, library screening, cDNA subtraction and microarray subtraction assays. This Kit is also suitable for the characterization of alternative splicing forms of eukaryotic mRNAs.

The Kit is ideal for the amplification and cloning of long sequences, owing to the inclusion of Bioline’s reagents BIO-X-ACT™ Long DNA Polymerase and Reverse Transcriptase. RiboSafe RNase Inhibitor is also included, to prevent RNA degradation, which is especially important for the analysis of rare transcripts.

Kit Components

Reverse Transcriptase
5x RT Reaction Buffer
3´ RACE Adaptor
DEPC-treated Water
Ultra-pure dNTPs
50mM MgCl2
RNase Inhibitor
BIO-X-ACT™ Long DNA Polymerase
10x OptiBuffer
3´ RACE Outer Reverse Primer
3´ RACE Nested Reverse Primer
pUC19 Cloning Vector

Overview

The 3’ RACE procedure is demonstrated in the schematic diagram (see product images). First strand cDNA synthesis is carried out with either total RNA or poly(A)-selected RNA, using the supplied 3’ RACE Adaptor and the Reverse Transcriptase.

PCR is then performed on the cDNA template using the supplied 3’ RACE Outer Reverse Primer,
BIO-X-ACT™ Long , Bioline’s high fidelity polymerase for long templates, and a forward primer (supplied by the user). A single amplification is usually sufficient; however, should the 3’ RACE reaction require further amplification, a 3’ RACE Nested Reverse Primer is provided.

PCR products can then be cloned into the supplied pUC19 cloning vector using the SbfI sites included in the 3’ RACE Reverse Primers and forward primer (supplied by the user). SbfI is an 8-base nucleotide recognition site, minimizing the chance of the cDNA being digested. For optimal ligation conditions we recommend using Quick-Stick Ligase (Cat No. BIO-27027).

Storage Conditions

The 3´ RACE Kit components can be stored for 6 months at -20°C.

Shipping Conditions

On Dry Ice or Blue Ice