Effect of Altered Plasmid Size on pBR322 Exclusion From pUC19/pBR322 Co-Transformants

Abstract

Size modified mutants of the plasmid vector pBR322 were constructed and transfected into E.coli DH5 ∝ cells. After isolating and purifying sufficient amounts of unmodified pBR322, samples were cut using sequential and double-digest restriction enzyme protocols. Cleaving the plasmid with the enzymes Sty I and Msc I made a small 86 bp deletion, and a larger 295 bp deletion was also made on a separate sample using the Kpn 2I and Sty I enzymes. The cut plasmids were blunt-ended and re-ligated, and transfected into E. coli DH5 ∝ via electroporation. Clones with the 86 bp deletion were successfully isolated, and the altered plasmids isolated from them. No clones were isolated that contained the 295 bp deletion.