Stem Cell cDNA Libraries

Human Embryonic Stem Cell and Stem Cell Line cDNA libraries are constructed 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 special vectors and transformed into T1 phage resistant E. coli.

Average insert size and insert size range are determined by restriction enzyme digestion of 24 clones picked at random from each library. The 4 kb BioExpress 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.