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Cancer Genome Characterization Initiative

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Library Info Page

Library ID

Library Name:Mouse P7-10 subtracted cDNA library containing dorsal root ganglion (DRG) enriched transcripts
Organism:Mus musculus
UniGene Library ID:24175
Tissue Description:
Library Keywords:EST

Clones and Sequences

#Clones Generated to Date:
#Sequences Generated to Date:1535

Library Preparation Details

Description:Poly(A)+ mRNA was purified from P7-10 wild type C57BL/6 mouse cerebellum and DRG and used to carry out cDNA library construction using Invitrogen's SuperScript Choice System for cDNA Synthesis. Blunt, double-stranded cDNAs were size-selected on an agarose gel and prepared for directional cloning in the Stratagene Lambda Zap-CMV XR vector (Agilent Technologies, Santa Clara CA) by sequential ligation to a 500-fold molar excess of EcoR1 adaptor and Xho1 digestion. Lambda ZAP-CMV cDNA ligations were packaged into lambda particles using Gigapack III Gold Packaging extracts (Stratagene). Mass library-wide excision of the phagemid resident in the lambda library was carried out using reagents and protocols provided by the Lambda Zap-CMV XR vendor (Stratagene). The mass-excision process yielded a pCMV-Script EX mammalian-expression phagemid library. The excised mammalian expression libraries in pCMV-Script EX were used to carry out subtractions (DRG - cerebellum) essentially as previously described (Bonaldo et al., 1996). Subtracted hybridizations were processed by Hydroxyapatite (HAP) chromatography to remove double-stranded cDNAs resulting from hybridization of DRG single-stranded circles to complementary cerebellum PCR products. DRG single-stranded circles with no complement in the cerebellar PCR product population remain single-stranded, are not retained by the HAP column and represent mRNA species expressed either exclusively or predominantly in DRG. Recovered single-stranded circles that do not adhere to the HAP column were desalted, concentrated and used in a primer extension reaction (T3 primer) to generate partially double-stranded plasmids fit for electroporation. The partially double-stranded plasmid population was electroporated into DH10B bacterial cells to generate the subtracted library.
R. Site1:EcoRI
R. Site2:XhoI
Lab Host:
Vector:pCMV-Script EX
Vector Type:Plasmid
Tissue Supplier:
Library Producer: