Cloning of poly-d(A:T) duplex in E. coli

Abstract

Plasmid POTC-10 DNA which is a derivative of PBR322 and contains trp-E gene promoter of $\underline{E}$. $\underline{coli}$ was isolated from E. coli HB101. This plasmid DNA was digested with ClaI restriction enzyme and treated with bacterial alkaline phosphatase to inhibit self-ligation. Poly-d(A:T) duplex (about 1 kilo-base-pair) was purchased from P.L. Biochem.. Because poly-d(A:T) duplex was very heterogeneous in molecular size, it was denatured and renatured. ClaI-linker was labelled with $r-^{32}$p ATP. This labelled ClaI-linker was ligated to poly-d(A:T) duplex with $T_4$-DNA ligase and digested with ClaI restriction enzyme. The ClaI digested POTC-10 plasmid DNA and poly-d(A:T) duplex:ClaI-linker were ligated with $T_4$-DNA ligase. This ligate was used to transform $\underline{E}$. $\underline{coli}$ HB101 competent cell treated with $CaCl_2$ buffer solution. Plasmid DNA was isolated from the each transformant (ampcilline plate) by rapid small scale isolation method. Their size was checked on 1% agarose gel electrophoresis and about 15% of the isolated DNA was appeared to have increased size. These plasmid DNA which showed an increase in length were digested with Hind III restriction enzyme and checked on 1% agarose gel electrophoresis whether they were the dimer of POTC-10 or not. From this, it was certain that they may be not the dimer but the recombinant which were inserted with 1 kilo-base paired fragment. Then the recombinant DNA were digested with ClaI restriction enzyme and the existence of poly-d(A:T) duplex was checked by colony hybridization and by southern blotting hybridization technique.