Takara Clontech’s Tips for cDNA Synthesis and Library Construction

27 Nov 2014

Kerry Parker

CEO

Editorial article

Takara offers a range of SMART (Switching Mechanism at 5’ End of RNA Template) solutions for cDNA library construction and synthesis, with kits designed specifically for your starting material.

RNA-Seq with well-represented 5’ end sequences: A common problem with conventionally-generated cDNA libraries is the high percentage of 5'-truncated cDNAs, due to partially degraded starting mRNA and incomplete first-strand synthesis. The SMART technology ensures uninterrupted cDNA synthesis, creating cDNAs with well-represented 5’ end sequences.

1. For cDNA synthesis from high quality RNA or cells, use the SMARTer Ultra Low Input RNA Kit for Sequencing. This kit is designed to produce cDNA libraries for RNA-seq directly from single cells or very low-input total RNA. The kit generates full-length cDNA libraries from as few as 1–1,000 cells, or as little as 10 pg–10 ng of total RNA. An example study of cDNA synthesis from a single cell can downloaded here.
2. For cDNA synthesis from laser capture microdissection (LCM) samples, formalin-fixed, paraffin-embedded (FFPE) tissue, prokaryotic samples, or samples with degraded RNA, try the SMARTer Stranded RNA-Seq Kits. These kits offer a solution for generating libraries for Illumina® sequencing platforms. Again, the kit can be used with a very low amount of starting material (100 pg to 100 ng of RNA).
3. To synthesize cDNA from high input total RNA samples of any quality, the SMARTer Stranded Total RNA Sample Prep Kit seamlessly blends efficient rRNA removal and strand-specific library generation in around five hours.

Cloning of full-length cDNA: The SMART cDNA library construction kits enable the cloning of full-length cDNA, as demonstrated in this recent application note. As shown in this article, SMART libraries contain a higher percentage of full-length clones than libraries constructed by conventional methods or other full-length cDNA synthesis protocols. The SMART libraries can also be produced from as little as 50 ng of total RNA.

Insert Your Library Into any Vector: Once your cDNA library is constructed, you can screen the library using PCR to determine how successful the library construction has been, and isolate your target gene(s), to be inserted into an expression vector. In-Fusion Cloning is designed to join fragments of DNA with 15 complementary bp at their ends. In-Fusion kits can be used to precisely transfer your SMARTer cDNA into any linearized vector. The In-Fusion SMARTer Library Construction protocol can be completed in just a few steps due to a highly efficient cDNA synthesis and cloning process. Only three enzymes are required to complete the entire protocol, as opposed to the usual 6-8 enzymes required for other methods.

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