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ExactSTART™ Small RNA Cloning Kit

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ExactSTART™ Small RNA Cloning Kit

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产品名称:

ExactSTART™ Small RNA Cloning Kit

货号
ES81005
规格
5 Rxns
存储温度
-70℃
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产品描述

Applications

  • Generation of cloned libraries derived from small cellular RNAs.

The ExactSTART™ Small RNA Cloning Kit* generates a cloned library derived from small RNA transcripts, starting with a total RNA sample. The kit selectively tags and converts small RNAs that contain 5´-monophosphate/ 3´-hydroxyl ends (5´ pN—OH 3´) or 5´-triphosphate/3´-hydroxyl ends (5´ pppN—OH 3´) to amplified dsDNA. Species of RNA tagged by the kit include miRNA, other small noncoding RNAs, and small primary transcripts. The amplified dsDNA is ligated into the pCDC-1K™ Cloning-Ready Vector (provided in the kit) and then transformed into a competent E. coli host (supplied by the user).

An optimized RNA ligation reaction is used to tag the 5´ end, and cDNA synthesis is used to tag the 3´ end of the RNAs (Fig. 1). An optional step, using a unique RNA 5´ Polyphosphatase* enzyme (discovered and characterized by EPICENTRE scientists), enables tagging and dsDNA synthesis of small primary transcripts with 5´-triphosphorylated ends. An initial, simple RNA size-selection procedure removes large RNAs such as mRNA, and 18S and 28S rRNA, to ensure that only small RNAs are converted to dsDNA and subsequently cloned.

Note: Plant miRNAs cannot be cloned with this kit, due to the 2´-O-methyl group at the 3´ end of the RNA.

Benefits

  • Strong selection process for cloning of miRNAs and other small noncoding RNAs and primary transcripts with 5´-monophosphorylated or 5´-triphosphorylated ends (Table 1).
  • Fast reactions: Clone and transform cells in 2 days.
  • Includes pCDC-1K™ Cloning-Ready Vector (linearized and dephosphorylated).

 

Table 1. Identification of characterized human miRNAs in a HeLa cDNA library prepared using the ExactSTART™ Small RNA Cloning Kit. Ninety-six randomly selected clones from the library were analyzed by sequencing.

miRNA Hits
hsa-let-7a 1
hsa-let-7i 1
hsa-miR-15b 1
hsa-miR-18a 1
hsa-miR-19b 2
hsa-miR-20a 2
hsa-miR-21 7
hsa-miR-23a 1
hsa-miR-24 2
hsa-miR-26a 1
 
miRNA Hits
hsa-miR-27b 1
1 hsa-miR-92a 7
hsa-miR-96 1
hsa-miR-99a 1
hsa-miR-125a-5p 1
hsa-miR-181a 1
hsa-miR-196a 1
hsa-miR-221 1
hsa-miR-365 1
hsa-miR-484 1

 

 

Figure 1 (click to enlarge). Schematic overview of the process for the ExactSTART™ Small RNA Cloning Kit.


*Patent pending.

 

 Catalog No.
  Size

ExactSTART™ Small RNA Cloning Kit
   ES81005 5 Reactions
Contents: RNA 5´ Polyphosphatase and Buffer, Poly(A) Polymerase and Buffer, Glycogen, RNase Inhibitor, ATP, T4 RNA Ligase and Buffer, 10X Ligase and RT Buffer, Small RNA Acceptor Oligo, RNase Solution, MMLV-RT, Small RNA cDNA Synthesis Primer, Small RNA PCR Primer 1, Small RNA PCR Primer 2, FailSafe™ 2X PreMix E, Small RNA Enrichment Solution, Nuclease-Free Water, DTT, Control RNA Template, Fast-Link™ 10X Ligase Buffer, Fast-Link™ DNA Ligase, Cloning Vector.

 

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