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cDNA Library Construction Services

Total RNA and mRNA Preparation

Catalog #: 10000 (Total RNA purification from tissue/cells; 10001 (mRNA purification from tissue/cells); 10002 (mRNA purification from total RNA)

The production of a high quality library containing a large number of full-length cDNA clones requires that the tissue and RNA preparation be performed in a certain way. These methods will help to guarantee that the starting material and ultimately the cDNA library are not compromised.

To isolate total RNA from frozen tissue, the tissue is ground to a fine powder using liquid nitrogen and a mortar and a pestle. To isolate RNA from cells, the cells are pelleted and the pellet washed with PBS. Total RNA is quickly isolated by homogenization or lysis using a reagent, such as TRIzol or equivalent. Total RNA is further processed to mRNA using poly A+ selection. Total RNA and mRNA are dissolved in nuclease-free water (no DEPC). We provide quality controlled RNA from customer supplied materials.

Quality Testing:

The A260/A280 ratio of the RNA must be at least 1.8.

Gel analysis of the total RNA must show that the 28S rRNA band is nearly twice the intensity of the 18S rRNA band.

Tissue and RNA Preparation Suggestions

cDNA Library Amplification

Catalog #: 10003

This is a method that we use to expand the primary or normalized library by gentle culture in a semi-solid agar matrix, similar to culture on agar plates. This method minimizes any effects on the cDNA representation that would otherwise occur in a typical liquid culture. Generally, we can amplify 1×107 primary clones 10,000 fold to 1×1011 colony forming units.

Standard cDNA Libraries

Catalog #:10004

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 3×106 primary clones with ≥87% recombinant clones containing an average insert size of at least 1kb, however, we generally produce libraries of 10×106 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 1×108), 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.

Characteristics of a cDNA Library Constructed From a Standard Amount of HeLa mRNA

mRNA (ng) Total # of primary clones Average insert size (kb)* Percent recombinants Insert size range (kb)*
5,000 12.0×107 2.24 96 0.5 – 4.4

mRNA (ng), Total # of primary clones, Average insert size (kb)*, Percent recombinants, Insert size range (kb)**Average insert size and insert size range determined by restriction enzyme digestion of 24 clones picked at random from each library.

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.

Tissue and RNA Preparation Suggestions

Large Insert cDNA Libraries

Catalog #:10005

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 1×106 primary clones with ≥87% recombinant clones containing an average insert size of at least 3kb, however, we generally produce libraries of 3×106 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 2×108), 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.

Characteristics of a cDNA Library Constructed From Large Transcripts of HeLa mRNA

mRNA (ng) Total # of primary clones Average insert size (kb)* Percent recombinants Insert size range (kb)*
5,000 2.1×107 4.26 96 1.0 – 6.2
2,000 2.5×106 4.46 100 2.4 – 7.2

mRNA (ng), Total # of primary clones, Average insert size (kb)*, Percent recombinants, Insert size range (kb)**Average insert size and insert size range determined by restriction enzyme digestion of 24 clones picked at random from each library.

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.

Tissue and RNA Preparation Suggestions

Microquantity cDNA Libraries

Catalog #:10006

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 1×106 primary clones with ≥87% recombinant clones containing an average insert size of at least 1kb, however, we generally produce libraries of 3×106 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 1×107), 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.

Characteristics of a cDNA Library Constructed From a Microquantity Amount of HeLa mRNA

mRNA (ng) Total # of primary clones Average insert size (kb)* Percent recombinants Insert size range (kb)*
2,000 7.3×107 2.43 100 1.7 – 5.5
1,000 8.0×106 2.43 100 0.9 – 5.2
500 6.7×105 1.75 100 0.8 – 2.9

mRNA (ng), Total # of primary clones, Average insert size (kb)*, Percent recombinants, Insert size range (kb)**Average insert size and insert size range determined by restriction enzyme digestion of 24 clones picked at random from each library.

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.

Tissue and RNA Preparation Suggestions

Nanoquantity cDNA Libraries

Catalog #:10007

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 2×105 primary clones with ≥87% recombinant clones containing an average insert size of at least 800 bp, however, we generally produce libraries of 5×105 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.

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 7.2×107 1.20 100 0.5 – 3.2
50 1.5×107 1.35 100 0.6 – 3.5
10 1.7×106 1.00 100 0.4 – 2.9
5 9.0×105 1.01 96 0.4 – 3.0
2 2.0×105 0.74 94 0.2 – 2.3
1 4.5×104 0.92 94 0.2 – 3.6

mRNA (ng), Total # of primary clones, Average insert size (kb)*, Percent recombinants, Insert size range (kb)**Average insert size and insert size range determined by restriction enzyme digestion of 24 clones picked at random from each library.

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.

Tissue and RNA Preparation Suggestions

Normalized cDNA Libraries

Catalog #: 10008

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 1×106 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 3×106 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 and RNA Preparation Suggestions

Subtracted cDNA Libraries

Catalog #:10009

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 1×105 subtracted clones with > 87% recombinant clones containing an average insert size of at least 1 kb. However, we generally produce libraries of >5×105 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 subtacted 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 –80°C is recommended.

Tissue and RNA Preparation Suggestions

454-Ready cDNA

Catalog #:10030

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 subtrate 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.

PCR and RT-PCR Services

Catalog #: 10040 (PCR/RT-PCR Detection and Identification); 10041 (PCR/RT-PCR Large Scale Amplification); 10042 (PCR/RT-PCR Cloning)

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.