clontech试剂盒 SMARTer_RACE 5'/3' Kit User Manual
insert, analyze the DNA by PCR screening (with your GSPs) or restriction digest (with EcoRI and
HindIII, which flank the cloning site). NOTE: For 5’-RACE products, we recommend picking at least 8–10 different independent clones in
order to obtain the maximum amount of sequence at the 5’end (see the note on full-length cDNA in
Section C, below).
C. Sequencing RACE Products
Once you have identified the clones containing the largest gene-specific inserts, obtain as much sequence
data as you can. Ideally, you will be able to sequence the entire open reading frame, as well as the 5’ and
3’ untranslated regions. NOTE: The provided pRACE vector is a pUC19-based vector, and is compatible with M13 sequencing
primers for characterization of your cloned insert(s). Because In-Fusion cloning is directional, you can
preferentially use the M13F primer to sequence into the UPM end, and the M13R primer to sequence into
the gene-specific end.
The UPM contains a T7 priming site which can be used for Sanger sequencing, but we recommend using
M13 primers to get full clean reads of your experimental sequence. The T7 priming sites are too close to
the 5’- and 3’-cloning sites to ensure complete coverage in the sequencing trace.
A note on full-length cDNA
No method of cDNA synthesis can guarantee a full-length cDNA, particularly at the 5’ end. Determining
the true 5’ end requires some combination of RNase protection assays, primer extension assays, and
cDNA or genomic sequence information. Many SMARTer RACE cDNAs include the complete 5’ end of
the cDNA; however, severe secondary structure may block the action of RT and/or SeqAmp DNA
Polymerase in some instances. In our experience, SMARTer RACE products and full-length cDNAs
compare favorably in this regard with cDNAs obtained by conventional RACE or from libraries.
Options for generating full-length cDNA
After the RACE products have been characterized by partial or complete sequencing, you can generate
the full-length cDNA by one of two methods:
By long distance PCR (LD PCR) using primers designed from the extreme 5’ and 3’ ends of your
cDNA and the 5’-RACE-Ready cDNA as a template.
By cloning overlapping 5’- and 3’-RACE fragments using a restriction site in the overlapping region
(if available). If no suitable restriction sites are available, you can alternately design new GSPs
suitable for multi-fragment In-Fusion cloning.