Full Length Enriched cDNA Synthesis
MINT-2 cDNA Synthesis Kit from Evrogen
Evrogen´s Mint-2 cDNA synthesis kit is designed to synthesize full-length-enriched double stranded (ds) cDNA from as little as 250 ng Total or 100 ng poly(A)+ RNA
• Fast cDNA synthesis protocol
• High content of full length transcripts
• Optimized for various downstream applications, including next generation sequencing
• Free Encyclo PCR kit (see link below) included
• Construction of cDNA libraries
• Subtractive Hybridization (SSH)
• cDNA normalization using Evrogen TRIMMER kit/Duplex Specific Nuclease (see link below)
• High-throughput Sequencing on the Next Generation Sequencing platforms (Roche/454, ABI/SOLiD or Illumina/Solexa)
• And other applications like virtual Northern Blots or array probe generation
Mint-2 cDNA synthesis kit is based on a novel technology (patent pending) utilizing the specific features of MMLV-based reverse transcriptase (RT). First strand cDNA synthesis starts from the 3´-end CDS adapter containing an oligo(dT) sequence that anneals to poly(A) stretches of RNA. When Mint RT reaches the 5´-end of the mRNA, it adds several non-template nucleotides, primarily deoxycytidines, to the 3´-end of the newly synthesized first-strand cDNA (Schmidt and Mueller, 1999). This oligo(dC) stretch base pairs to complementary oligo(dG) sequence located at the 3´-end of a special deoxyribooligonucleotide adaptor, called PlugOligo. Mint RT identifies PlugOligo as an extra part of the RNA-template and continues first strand cDNA synthesis to the end of the oligonucleotide, thus incorporating PlugOligo sequence into the 5´-end of cDNA.
The last 3´-dG residue of the PlugOligo is a terminator nucleotide containing a 3´-phosphate group. This blocking group prevents unwanted extension of the PlugOligo. Under standard conditions Mint RT can hardly use PlugOligo as a template; however, a specially tailored IP-solution (solution for incorporation of PlugOligo sequence) dramatically increases the efficiency of this process.
At the final step, ds cDNA is amplified by PCR. Use of Encyclo polymerase (see link below) and specially designed primers allows synthesis of full-length-enriched cDNA that is flanked by PlugOligo and CDS adapter sequences.
Applicable to all eukaryotic organisms and all tissue sources
Figure 2. MINT-amplified cDNA from different sources: 1 - mouse liver; 2 - mouse skeletal muscle; 3 - mouse brain; 4 - human leucocytes; 5 - human lung; 6 - human skeletal muscle; 7 - mosquito grub; 8 - copepod Pontella sp.; 9 - tomato Lycopersicon esculentum. M - 1 kb DNA size marker.
Compatible with TRIMMER cDNA normalization kit
cDNA prepared with the MINT-2 kit can be normalized using TRIMMER Kit (Cat# NK001-EV)- see link below- to decrease the prevalence of high abundant transcripts.
Schmidt WM and Mueller MW (1999) CapSelect: a highly sensitive method for 5´ CAP-dependent enrichment of full length cDNA in PCR-mediated analyses of mRNAs. Nucleic Acid Res. 27, e31.
Diachenko L, Lau YF, Campbell AP, Chenchik A, Mogadam F, Huang B, Lukyanov S, Lukyanov K, Gurskaya N, Sverdlov ED, Siebert D. (1996) Suppression Subtracive Hybridization: A method for generating differentially regulated or tissue-specific cDNA probes and libraries. Proc. Natl. Acad. Sci. USA 93(12): 6025-6030. / PMID:8650213
Franz, O., Bruchhaus, I.I, Roeder, T. (1999) Verification of differential gene transcription using virtual northern blotting. Nucleic Acids Res. 27: e3.
Product Use Limitations: MINT Kit is intended for research purposes only.
Endnotes: PCR is the subject of patents issued in certain countries. The purchase of this kit does not include a license to perform PCR. However, many researchers may not be required to obtain a license. Other investigators may already have a license to perform PCR through use of a thermal cycler with the appropriate label license.
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|Mint-2 Full Length Enriched cDNA Synthesis Kit (for constructing cDNA libraries, subtractive hybridization (SSH), cDNA normalization using Evrogen Trimmer kit, and high-throughput sequencing on the next generation sequencing platforms)||SK005-EV||20 rxns||536 €||DETAILS||Add to Cart|