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Cancer Genome Characterization Initiative

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Library Info Page


Library ID

Library Name:Kaestner ngn3 - - subtracted
Organism:Mus musculus
Strain:129/Sv x CD1
UniGene Library ID:10349
Tissue Description:
Library Keywords:fetus, pancreas, normal, bulk, subtracted, EST, plasmid vector, crossbred

Clones and Sequences

#Clones Generated to Date:
#Sequences Generated to Date:1165

Library Preparation Details

Description:Both wildtype (target) and ngn3-/- (driver) cDNA libraries were amplified. The ssDNA from the wildtype cDNA library was prepared by infecting with M13KO7 and blocked with 5'GCGGCCGCT(15) oligo before hybridization. SalI digested dsDNA from the ngn3-/- cDNA library was used to synthesize biotinylated RNA with T7 RNA polymerase. Subtractive hybridization was performed at 42oC in 80% formamide, 100 mM HEPES pH 7.5, 2mM EDTA and 0.2% SDS. Streptavidin was used to remove clones common between the two libraries. After repair of the subtracted ssDNA with 5'GCGGCCGCT(15) oligo using Taq polymerase and Vent polymerase, the subtracted cDNA library was digested with SpeI and electroporated into DH12S cells (LTI).
R. Site1:Not I
R. Site2:Sal I
Lab Host:DH12S
Vector:pSPORT1 (GIBCO)
Vector Type:Plasmid (Amp resistant)
Tissue Supplier:
Library Producer: