BioSample

Poly(A)+RNA was isolated from 58 ug of total RNA by one round of oligo(dT) selection with oligo(dT) coated magnetic particles (Seradyn, Inc.). From 1.2 ug of this poly(A)+RNA mRNA, a cDNA library was constructed by using an oligo dT primer-adapter containing a Not I site and Moloney Murine Leukemia Virus Reverse Transcriptase (M-MLV RT) to prime and synthesize first strand cDNA. After the second strand was synthesized, the double stranded cDNA was digested with Not I, size fractionated (>1.4 kb) and cloned directionally into the Not I and Sma I sites of the pCS111 vector. From one bulk ligation (300 ng of pCS111 vector, Not I-Sma I cut, and 120 ng of Not I digested cDNA per 120 ul of ligation), followed by electroporation into T1 phage resistant E. coli, 1.2 x 107 primary clones were produced. The cDNA inserts from 24 randomly picked clones were sized by digestion with Not I and Eco RI. The average insert size of these 24 clones was 1.9 kb.