clontech试剂盒 SMARTer_RACE 5'/3' Kit User Manual
B. Additional Considerations for Design
The primers shown in Figure 3 will create overlapping 5’- and 3’-RACE products. If a suitable restriction
site is located in the region of overlap, the fragments can subsequently be joined by restriction digestion
and ligation to create the full-length cDNA. If no suitable restriction sites are available, you can
alternately design new GSPs suitable for multi-fragment In-Fusion cloning. By designing primers that
give a 100–200-bp overlap in the RACE products, you will also be able to use the primers together as an
internal positive control for the PCR reactions. However, it is not absolutely necessary to use primers that
give overlapping fragments. In the case of large and/or rare cDNAs, it may be better to use primers that
are closer to the ends of the cDNA and therefore do not create overlapping fragments. The primers
themselves can overlap (i.e., be complementary).
C. Location of Primer Sequences within Genes
We have had good success using the SMARTer RACE Kit to amplify 5’ and 3’ cDNA fragments that
extend up to 6.5 kb from the GSP binding sites. Nevertheless, for optimum results, we recommend
choosing your primers so that the 5’- and 3’-RACE products will range from 1–3 kb in length. If you are
working with an annotated genome, we suggest using NCBI’s Primer-BLAST to aid in your design for
each transcript.
D. Nested Primers
We recommend that you do not use nested PCR in your initial experiments. The UPM Primer and a GSP
will usually generate a good RACE product with a low level of nonspecific background. However, nested
PCR may be necessary in some cases where the level of background or nonspecific amplification in the
5’- or 3’-RACE reaction is too high with a single GSP. In nested PCR, a primary amplification is
performed with the outer primers and, if a smear is produced, an aliquot of the primary PCR product is re-
amplified using the inner primers. The SMARTer RACE protocols include optional steps indicating
where nested primers can be used. The Universal Primer Short (provided with the kit) can be used for
both 5’- and 3’-RACE with nested primers.
Nested gene specific primers (NGSP) should be designed according to the same guidelines discussed
above. If possible, nested primers should not overlap with the outer gene-specific primers; if they must
overlap due to limited sequence information, the 3’ end of the inner primer should have as much unique
sequence as possible. Additionally, your nested primers should also contain the 15 bp overlap required for
In-Fusion cloning.