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cDNA Library Construction Services Insight Biotechnology is pleased to offer innovative, high quality cDNA library services from Express Genomics. Express Genomics is a spinout company founded by former employees of Life Technologies, who’s staff had years of experience delivering high quality cDNA library services. The milestones made in mapping the human, mouse and rat genomes has resulted in a growing need to more rapidly discover and express novel genes from diverse tissues and species. To meet this demand, Express Genomics have developed innovative technology to produce libraries containing more full-length cDNA inserts. Other services they offer include libraries with an average insert size of 3kb, libraries produced from small amounts of starting tissue and RNA, normalized and subtracted libraries. Express Genomics will construct cDNA libraries from customer supplied tissue, cells or RNA. Insight Biotechnology will deliver libraries within 4 to 6 weeks after tissue, cells or RNA is received and after the order is accepted. However, to ensure that we understand the goals of your project and when to expect your samples, please DO NOT send materials until we contact you. High Quality 454-Ready cDNAHigh Quality 454-Ready cDNA is made from our custom or catalog cDNA libraries. Briefly, library DNA is digested with Not I and in vitro RNA transcripts are produced using the SP6 RNA polymerase promoter. Then, first strand cDNA is made from these transcripts using a modified primer adapter that reduces the size of the poly A sequence (to about 20 As). After the second strand is synthesized, the double stranded (ds) cDNA is blunt ended and size fractionated. This ds cDNA is now ready to be used with the 454 sequencing method or with other next generation sequencing technology (i.e., illumina). We guarantee at least 6 µg of ds cDNA, 150 ng/µl concentration in TE and 1 kb average cDNA size, however, we generally produce 8 µg of cDNA, 200 ng/µl concentration and 1.5 kb average cDNA size. We will make 454-ready cDNA from our custom or catalog libraries or directly from customer supplied cDNA libraries, tissue or RNA.
Quality testing: Gel analysis of cDNA population. Shipment and storage conditions: 454-ready cDNA will be shipped in dry ice. Storage of cDNA at -80°C is recommended. Tissue & RNA Preparation Suggestions Contact Insight Biotechnology’s scientific team to discuss this technology in greater depth and receive a quote for custom services: Tel +44(0)208 385 0303 or Email:info@insightbio.com PCR and RT-PCR ServicesWe offer the following PCR and RT-PCR services on customer supplied material.
We will work with you on all parts of your project from the design of the oligonucleotides through the analysis, quantitation, or cloning of your PCR products. A detailed certificate of analysis will be provided to you when your project is completed. Tissue & RNA Preparation Suggestions Contact Insight Biotechnology’s scientific team to discuss this technology in greater depth and receive a quote for custom services: Tel +44(0)208 385 0303 or Email:info@insightbio.com High Quality Standard cDNA LibrariesHigh Quality Standard cDNA Libraries are produced using our proprietary technology. First strand cDNA is made from mRNA that is primed using oligo dT. After the second strand is synthesized, the double stranded cDNA is size fractionated, cloned directionally into our Express I vector and transformed into T1 phage resistant E. coli. We guarantee at least 3x106primary clones with ≥87% recombinant clones containing an average insert size of at least 1kb, however, we generally produce libraries of 10x106 primary clones with >95% recombinant clones containing ≥1.5kb average insert size. We will make standard libraries from customer supplied tissue (≥1 g), cells (at least 1x108), total RNA (≥1 mg) or mRNA (≥5 µg). We provide quality controlled primary or amplified libraries.
Quality testing: Percentage of recombinant clones and average insert size determined by gel analysis of 24 clones picked at random. Shipment and storage conditions: Libraries will be shipped in dry ice. Storage of libraries at -80°C is recommended. Characteristics of a cDNA Library Constructed From a Large Insert Amount of HeLa mRNA
*Average insert size and insert size range determined by restriction enzyme digestion of 24 clones picked at random from each library. Tissue & RNA Preparation Suggestions Contact Insight Biotechnology’s scientific team to discuss this technology in greater depth and receive a quote for custom services: Tel +44(0)208 385 0303 or Email:info@insightbio.com High Quality Large Insert cDNA librariesHigh Quality Large Insert cDNA Libraries are constructed using our state-of-the-art methods for cDNA synthesis and size fractionation to produce libraries containing the largest cDNA inserts available. Although these libraries are made using a different process, they share the same advantageous features as our standard libraries (directional cloning into Express I and T1 phage resistant E. coli). We guarantee at least 1x106 primary clones with ≥87% recombinant clones containing an average insert size of at least 3kb, however, we generally produce libraries of 3x106 primary clones with >95% recombinant clones containing ≥4kb average insert size. We will make large insert libraries from customer supplied tissue (≥2 g), cells (at least 2x108), total RNA (≥2 mg) or mRNA (≥10 µg). We provide quality controlled primary or amplified libraries. - High quality size fractionated cDNA. Characteristics of a cDNA Library Constructed From a Large Insert Amount of HeLa mRNA
Tissue & RNA Preparation Suggestions Contact Insight Biotechnology’s scientific team to discuss this technology in greater depth and receive a quote for custom services: Tel +44(0)208 385 0303 or Email:info@insightbio.com High Quality MicroQuantity cDNA LibraryHigh Quality Microquantity cDNA Libraries are constructed using our state-of-the-art methods for cDNA synthesis and cloning to produce libraries from small amounts of starting material. Like our standard libraries, these libraries are directionally cloned into Express I and then transformed into T1 phage resistant E. coli. We guarantee at least 1x106 primary clones with ≥87% recombinant clones containing an average insert size of at least 1kb, however, we generally produce libraries of 3x106 primary clones with >95% recombinant clones containing ≥1.5kb average insert size. We will make microquantity libraries from customer supplied tissue (<1g), cells (at least 1x107), total RNA (≥50 µg) or mRNA (≥500 ng). We provide quality controlled primary or amplified libraries. Shipment and storage conditions: Libraries will be shipped in dry ice. Storage of libraries at -80°C is recommended. Characteristics of a cDNA Library Constructed From a Large Insert Amount of HeLa mRNA
Tissue & RNA Preparation Suggestions Contact Insight Biotechnology’s scientific team to discuss this technology in greater depth and receive a quote for custom services: Tel +44(0)208 385 0303 or Email:info@insightbio.com High Quality NanoQuantity cDNA LibraryRecently, we developed methods that enable us to produce High Quality Nanoquantity cDNA Libraries from such small amounts of tissue and cells that material prepared by laser capture microdissection (LCM) or FACS can be used. Like our standard libraries, these libraries are directionally cloned into Express I and then transformed into T1 phage resistant E. coli. We guarantee at least 2x105 primary clones with ≥87% recombinant clones containing an average insert size of at least 800 bp, however, we generally produce libraries of 5x105 primary clones with >95% recombinant clones containing ≥1.0kb average insert size. We will make nanoquantity libraries from customer supplied tissue (at least 250 µg), cells (at least 25,000), total RNA (≥250 ng) or mRNA (≥5 ng). We provide quality controlled primary or amplified libraries.
Quality testing: Percentage of recombinant clones and average insert size determined by gel analysis of 24 clones picked at random. Shipment and storage conditions: Libraries will be shipped in dry ice. Storage of libraries at -80°C is recommended. Characteristics of a cDNA Library Constructed From a Nanoquantity Amount of HeLa mRNA
*Average insert size and insert size range determined by restriction enzyme digestion of 24 clones picked at random from each library. Tissue & RNA Preparation Suggestions Contact Insight Biotechnology’s scientific team to discuss this technology in greater depth and receive a quote for custom services: Tel +44(0)208 385 0303 or Email:info@insightbio.com High Quality Normalized cDNA LibrariesHigh Quality Normalized cDNA Libraries are produced using our primary cDNA libraries. Biotinylated driver cRNA produced from the T7 RNA polymerase promoter and single-stranded (ss) target cDNA produced from the F1 ori are hybridized to each other at a low Cot (concentration of driver times the time of hybridization) value. The cRNA:cDNA hybrids are removed by phenol extraction and the remaining ss target cDNA is converted to double-stranded DNA (dsDNA) with a repair oligo and Taq DNA polymerase. After electroporation of the dsDNA into T1 phage resistant E. coli, normalized clones are produced. The normalization process reduces the level of abundant and moderately abundant genes and enriches for rare genes. To determine the reduction of an abundant gene, we compare the relative representation of the actin gene in the primary library with the normalized library by hybridization of different dilutions of the primary and normalized dsDNA with an actin probe. Using this method, we determine that the actin gene is reduced at least 20 fold in the normalized library. We guarantee at least 1x106 normalized clones with > 87% recombinant clones containing an average insert size of at least 1 kb for standard normalized libraries and 3 kb for large insert normalized libraries. However, we generally produce libraries of 3x106 normalized clones with >95% recombinant clones containing an average insert size of 1.5 kb average insert size for standard normalized libraries and 4 kb for large insert normalized libraries. Features list:
Quality testing: Percentage of recombinant clones and average insert size determined by gel analysis of 24 clones picked at random. Actin gene reduction determined by hybridization of primary and normalized library. Shipment and storage conditions: Libraries will be shipped in dry ice. Storage of libraries at -80°C is recommended. Tissue & RNA Preparation Suggestions Contact Insight Biotechnology’s scientific team to discuss this technology in greater depth and receive a quote for custom services: Tel +44(0)208 385 0303 or Email:info@insightbio.com
High Quality Subtracted cDNA LibrariesHigh Quality Subtracted cDNA Libraries are produced using our primary target and driver cDNA libraries. Biotinylated driver cRNA produced from the T7 RNA polymerase promoter and single-stranded (ss) target cDNA produced from the F1 ori are hybridized to each other at a high Cot (concentration of driver times the time of hybridization) value. The cRNA:cDNA hybrids are removed by phenol extraction and the remaining ss target cDNA is converted to double-stranded DNA (dsDNA) with a repair oligo and Taq DNA polymerase. After electroporation of the dsDNA into T1 phage resistant E. coli, subtracted clones are produced. The subtraction process removes genes that are in common (i.e., actin) between two different libraries (i.e., normal versus disease) and enriches for unique and highly induced genes. To determine the effectiveness of the subtraction process, we compare the relative representation of the actin gene in the primary library (target) with the subtracted library by hybridization of different dilutions of the primary and subtracted dsDNA with an actin probe. Using this method, we determine that the actin gene must be reduced at least 20 fold in the subtracted library. Generally, we see at least 100 fold reduction of the actin gene in the subtracted library. We guarantee subtracted libraries of at least 1x105 subtracted clones with > 87% recombinant clones containing an average insert size of at least 1 kb. However, we generally produce libraries of >5x105 subtracted clones with >95% recombinant clones containing an average insert size of 1.5 kb. Features list:
Quality testing: Percentage of recombinant clones and average insert size determined by gel analysis of 24 clones picked at random. Actin gene reduction determined by hybridization of primary and subtracted libraries. Shipment and storage conditions: Libraries will be shipped in dry ice. Storage of libraries at –80oC is recommended. Tissue & RNA Preparation Suggestions Contact Insight Biotechnology’s scientific team to discuss this technology in greater depth and receive a quote for custom services: Tel +44(0)208 385 0303 or Email:info@insightbio.com Products and PricesPlease see a product listing for Express Genomics. Contact Insight Biotechnology for additional technical and pricing information: Tel +44(0)208 385 0303 or Email:info@insightbio.com
Contact Insight Biotechnology’s scientific team to discuss this technology in greater depth and receive a quote for custom services: Tel +44(0)208 385 0303 or Email:info@insightbio.com |
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