FINAL DRAFT University of California, Santa Cruz BIOL 101L

Naomi Schilpp Giulia Gurun BioL 101L 7 May 2019 Insertion of egfp Into pET41a-(+) to Create a Recombinant Plasmid Results: Ligation of egfp insert into pET41A-(+) vector A ligation reaction was used to create recombinant expression plasmid of pET41a- (+)/Enhanced Green Fluorescent Protein (egfp). Five ligations were performed, three of which were controls. The positive control contained only uncut pET41-a(+)/egfp recombinant plasmid DNA and sterile water. Two negative controls were done, one of which was only sterile water and the other was Nco/NotI cut pET41a(+) DNA, egfp insert DNA, sterile water, and ligase buffer but no DNA ligase. The two non-control ligations were 1:1 pET41A-(+): egfp molecular ratio (L1) and 1:3 pET41A-(+): egfp molecular ratio (L2) along with sterile water, ligase buffer, and DNA ligase. The positive control shows recombinant supercoiling with three bands; 5 kb, ~6 kb, and 10 kb (Figure 1, lane 4). The negative control with only water shows no bands as expected (Figure 1, lane 5). The negative control containing DNA but no ligase produced two bands; 6 kb and ~700 bp. L1 produced three bands; one above 10 kb, at 8 kb, and one at ~700 bp (Figure 1, lane 2). L2 shows three bands; one above 10 kb, at 8 kb, and one at ~700 bp (Figure 1, lane 3). This study source was downloaded by 2960