Preparation of cDNA library from jurkat-FHCRC cells

Abstract

The conditions for the production of interleukin-2(IL-2) from Jurkat-FHCRC cells were studied and poly(A$^+$) RNA were extracted from Jurkat cells stimulated with ConA and TPA. cDNAs were synthesized from the poly(A$^+$) RNA by two different methods. First, the ss-cDNAs were synthesized using an oligonucleotide of complementary sequence to the 3``-end of the IL-2 gene as a primer. One prominant band sized about 500 base pairs was reproducibly observed on the denaturing polyacrylamide gel and this was consistent with the size determined from the known sequence of the IL-2 gene. Second, cDNA library was synthesized using oligo(dT) as a primer. E.coli JM 83 strain was used for transformation. Approximately 50,000 white, ampicillin-resistant colonies were obtained on 5-bromo-4-chloro-3-indolyl-B-D-galactopyranoside (X-gal)-ampicillin plate. The insert sizes of cDNAs were ranged between 450 base pairs and 1,600 base pairs.