For the types of services we offer, please click on "Custom cDNA library services" under "Products" on the left. Please submit your specifications to info@helica.com or by calling 714-578-7830, and we will be happy to send you a price quote. Below is a summary of the most requested information regarding our cDNA library custom services.
Frequently Asked Questions
1) Q: What types of libraries do you offer?
A: "Presently, we offer several types of libraries including standard, microquantity, large insert, as well as, more customized libraries. We also have 80 different catalog libraries ready to ship. For instance, recently, we use a novel technology to make nanoquantity libraries. The terms standard, microquantity and nanoquantity refer to the amount of RNA used to construct the cDNA library. Standard libraries can be produced from at least 1 g of tissue, 1x108 cells, 1 mg of total RNA or 5 ?g of mRNA. Microquantity libraries can be produced from at least 100 mg of tissue, 1x107 cells, 50 ?g of total RNA or 500 ng of mRNA. Nanoquantity libraries can be produced from at least 100 ?g of tissue, 10,000 cells, 100 ng of total RNA and 1 ng of mRNA."
2) Q: How are these libraries made?
A: "These libraries are produced by using an oligo dT primer-adapter and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) to prime and synthesize first strand cDNA from mRNA. After the second strand is synthesized, the double stranded cDNA is size fractionated, cloned directionally into our Express 1 vector and transformed into T1 phage resistant E. coli. For our standard libraries, we guarantee at least 3x106 primary 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. For our microquantity libraries, 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. For our large insert size libraries, we guarantee at least 1x106 primary clones with ?87% recombinant clones containing an average insert size of at least 3kb, however, we generally produce large insert libraries of 3x106 primary clones with >95% recombinant clones containing ?4kb average insert size."
3) Q: What are the features of these libraries?
A: "The 4 kb Express 1 vector used for cloning is Puc based, confers ampicillin resistance and contains the CMV promoter for expression analysis. This vector also contains the SP6 and T7 RNA polymerase promoters flanking the MCS for RNA synthesis, the Amersham ET and M13 primer sites for sequencing and the F1 ori for single-stranded DNA production. By cloning the cDNA directionally into this vector the cDNA clones can be expressed, detected by antibody screening and the libraries can be used to produce normalized or subtracted libraries."
4) Q: Do you offer subtracted or normalized libraries? A: "Yes, the highest quality normalized and subtracted cDNA libraries in the market place. Our normalized and subtracted libraries have been sequence up to 50,000 clones deep by the Mammalian Genome Project (MGC), a section of the National Cancer Institute http://mgc.nci.nih.gov
and others. The results of the bioinformatics analysis demonstrated excellent gene diversity/complexity from our cDNA library construction technology. The pExpress 1 vector has a high copy number in bacteria and primes well in sequence reactions with average reads of over 800 bases. The libraries contain a high percentage of novel unidentified clones, a low rate of repetitive clones, as high as 74% full-length clones, and scored high marks as gene discovery tools.
5) Q: How quickly could you produce my library?
A: "We guarantee to deliver libraries within 4 to 6 weeks after tissue, cells or RNA is received and after the order is accepted. The acceptance of the order is based upon the quality of total RNA isolated from the tissue or cells. Indicators of total RNA quality are the A260/A280 ratio must be at least 1.8 and the 28S rRNA band is nearly twice the intensity of the 18S rRNA band by gel analysis."
6) Q: Would I be able to order the construction of a library that is not included in your catalog of custom libraries?
A: "Generally, the answer is yes. If you need other vectors, other cDNA synthesis methods (i.e., random priming), transformation into other E. coli cells, etc., we will work with you to reach your project goals."
7) Q: What is an amplified library?
A: "This is a method to expand the primary 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 1x107 primary clones 10,000 fold to 1x1011 colony forming units."
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