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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 cDNA

High 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.
 Features list:

  • High quality size fractionated cDNA
  • Reduced poly A size to improve sequencing results
  • Large amount of cDNA can be produced from nanoquantity levels of tissue or RNA
  • Ideal substrate for next generation sequencing methods.

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 Services

We offer the following PCR and RT-PCR services on customer supplied material.

  • Rapid detection and identification of contaminants in samples (i.e. bacterial, viral, etc.).
  • Large scale amplification to yield microgram to milligram amounts of PCR and RT-PCR products.
  • Cloning of PCR products into the vector of your choice.

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 Libraries

High 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.

Features list:

  • High quality size fractionated cDNA
  • Directionally cloned cDNA for expression, antibody screening, subtraction and normalization
  • Large numbers of primary clones
  • Low vector background

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


mRNA (ng)
Total # of Primary Clones Average Insert Size (kb)* Percent Recombinants Insert size range (kb)*
5000 12x107 2.24 96 0.5-4.4

*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 libraries

High 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.

Features list:

- High quality size fractionated cDNA.
- Directionally cloned cDNA for expression, antibody screening, subtraction and normalization.
- Large numbers of primary clones.
- Low vector background.

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


mRNA (ng)
Total # of Primary Clones Average Insert Size (kb)* Percent Recombinants Insert size range (kb)*
5000 2.1x107 4.26 96 1.0 - 6.2
2000 2.5x107 4.46 100 2.4 - 7.2

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 Library

High 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.

Features list:
- High quality cDNA clones.
- Directionally cloned cDNA for expression, antibody screening, subtraction and normalization.
- Large numbers of primary clones.
- Low vector background.

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


mRNA (ng)
Total # of Primary Clones Average Insert Size (kb)* Percent Recombinants Insert size range (kb)*
2000 7.3x107 2.43 100 1.7 - 5.5
1000 8.0x107 2.43 100 0.9 - 5.2
500 6.7x107 1.75 100 0.8 - 2.9

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 Library

Recently, 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.

Features list:

  • High quality cDNA clones
  • Directionally cloned cDNA for expression, antibody screening, subtraction and normalization
  • Large numbers of primary clones
  • Low vector background

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


mRNA (ng)
Total # of Primary Clones Average Insert Size (kb)* Percent Recombinants Insert size range (kb)*
100 7.2x107 1.20 100 0.5 - 3.2
50 1.5x107 1.35 100 0.6 - 3.5
10 1.7x106 1.00 100 0.4 - 2.9
5 9.0x105 1.01 96 0.4 - 3.0
2 2.0x105 0.74 94 0.2 - 2.3
1 4.5x104 0.92 94 0.2 - 3.6

*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 Libraries

High 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:

  • High quality size fractionated cDNA
  • Directionally cloned cDNA for expression, antibody screening, subtraction and normalization
  • Large numbers of normalized clones
  • Low vector background
  • Abundant and moderately abundant genes reduced and rare genes enriched in the normalized library

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 Libraries

High 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:

  • High quality size fractionated cDNA
  • Directionally cloned cDNA for expression, antibody screening, subtraction and normalization
  • Large numbers of subtracted clones
  • Low vector background
  • Unique and highly induced genes enriched in the subtracted library

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 Prices

Please 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


Catalog #
Item Name Price
Human
10101-N Brain Click Here
10102-N Fetal Brain Click Here
10106-N Liver Click Here
10107-N Lung Click Here
 
Human Embryonic Stem Cell Lines
MGC_279 HSF-1.14 Cell Line Click Here
MGC_281 HSF-6 Cell Line Click Here
     
Human Embryonic Stem Cell  
(see CoAs for specific developmental stage)
12259-N Normalized Click Here
12261-N Normalized Click Here
12263-N Normalized Click Here
 
Mouse
10104-N Brain Click Here
MGC_230 Thyroid Click Here
MGC_257 Oocyte Click Here
 
Rat
10103-N Brain Click Here
MGC_255 Brain Click Here
MGC_232 Lung Click Here
MGC_234 Heart Click Here
MGC_236 Kidney Click Here
MGC_238 Testis Click Here
MGC_253 Ovary Click Here
 
Rat Embryo
MGC_366 Brain Click Here
MGC_368 Liver Click Here
MGC_419 Kidney Click Here
MGC_421 Heart Click Here
   
Xenopus NICHD_XGC
(A mixture of Xenopus lavies and Xenopus tropicials)
XGC_Te2N Testis Click Here
XGC_FaBN Fat Bodies Click Here
XGC_Emb10 Stage9 Embryo Click Here
  Express Genomics Catalog cDNA Libraries
(NON-NORMALIZED)
Human
10101-A Brain Click Here
10102-A Fetal Brain Click Here
10105-A Heart Click Here
10106-A Liver Click Here
10107-A Lung Click Here
10108-A Prostate Click Here
10109-A Spleen Click Here
10110-A Placenta Click Here
10111-A Kidney Click Here
10112-A Testis Click Here
10113-A Thymus Click Here
 
Human Embryonic Stem Cell Lines
MGC_278 HSF-1.14 Cell Line Primary Click Here
MGC_280 HSF-6 Cell Line Primary Click Here
 
Human Embryonic Stem Cell
12258-P Primary Click Here
12260-P Primary Click Here
12262-P Primary Click Here
     
Monkey
10114-A Brain Click Here
     
Mouse
10104-A Brian Click Here
10115-A Thyroid Click Here
MGC_189 Thyroid Click Here
MGC_256 Oocyte Click Here
 
Rat
10103-A Brain Click Here
MGC_254 Brain Click Here
10116-A Lung Click Here
MGC_231 Lung Click Here
10117-A Testis Click Here
MGC_237 Testis Click Here
10118-A Heart Click Here
MGC_233 Heart Click Here
10119-A Kidney Click Here
MGC_235 Kidney Click Here
MGC_252 Ovary Click Here
 
Rat Embryo
MGC_365 Brain Click Here
MGC_367 Liver Click Here
MGC_418 Kidney Click Here
MGC_420 Heart Click Here
 
Xenopus NICHD_XGC
(A mixture of Xenopus lavies and Xenopus tropicials)
XGC_int(m) Embryonic Intestine Click Here
XGC_limb(m) Embryonic Hind Limb Click Here
XGC_skin(m) Embryonic Skin Click Here
XGC_tail(m) Embryonic Tail Click Here
XGC_tropInt54 Embryonic Small Intestine Stg 54 Click Here
XGC_tropInt60 Embryonic Sm Int Stg 60 Click Here
XGC_tropInt62 Embryonic Sm Int Stg 62 Click Here
XGC_tropInt63 Embryonic Sm Int Stg 63 Click Here
XGC_tropInt66 Embryonic Sm Int Stg 66 Click Here
XGC_bone Bone Click Here
XGC_tropEye1 Eye Click Here
XGC_limb Hind Limb Click Here
XGC_olfb Olfactory bulb Click Here
XGC_panc Pancreas Click Here
XGC_sple_PHA Spleen PHA-treated Click Here
XGC_thy Thymus Click Here
XGC_Te2 Testis Click Here
XGC_FaB Fat Bodies Click Here
XGC_Emb9 Stage9 Embryo Click Here
XGC_Emb9 Stage9 Embryo Click Here

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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