Ligation Lab Report

Ligation

The objective of this experiment was to ligate EGFP DNA inserts into pET41a(+) plasmids. A total of five ligations were performed, two actual ligations and three control ligations. The following reagents were utilized: Nco I/Not I cut pET-41a(+) DNA 50 ng/μL, EGFP cDNA insert 7 ng/μL, uncut pET-41a(+) DNA/EGFP recombinant plasmid DNA 25 ng/μL, ligase buffer 10X, and ligase.

To prepare ligation #1, a 1:1 molar ratio of pET41/EGFP, 2 μL digested pET41a(+) DNA, 1 μL EGFP cDNA insert, 14 μL sterile dH2O, 2 μL ligase buffer, and 1 μL DNA ligase were added to a micro centrifuge tube. To prepare ligation #2, a 1:3 molar ratio of pET41/EGFP, 3 μL digested pET41a(+) DNA, 1 μL EGFP cDNA insert, 12 μL sterile dH2O, 2 μL ligase buffer, and 1

Three bacterial cultures were received from laboratory staff. The colonies that cultures were grown from came from ligations and transformations that were prepared by the laboratory staff. The cultures prepared during the ligation and transformation steps were unusable due to unsuccessful ligation. Two colonies were taken from an LB/kanamycin/IPTG plate. One that fluoresced green under UV light, and one that did not fluoresce green under UV light. The third colony was taken from the LB/kanamycin plate, its ability to fluoresce is unknown. For each culture 1 mL of cell suspension was taken and added to a separate micro centrifuge tube. The cell suspensions were then centrifuged at 10,000 RCF for one minute to pellet the bacterial cells. The supernatant was then removed without disturbing the pellet. This process was repeated twice more until a total of 2.5 mL of cell suspension was removed from each culture and all the supernatant was removed from each micro centrifuge tube. The original protocol called for a total of 1.2 mL of cell suspension to be taken from each culture, but the laboratory staff determined that a greater volume was required in order to get a sufficient DNA concentration to be used for the DNA digestion. To each tube was added 200 μL Cell Suspension Solution, followed by vortexing until the pellets were completely re-suspended. Next 200 μL Cell Lysis Solution was added to each tube and then mixed

To the double digest tube was added 12 μL double digest master mix, and 2.5 μL sterile dH2O. To the single digest tube was added 11 μL single digest master mix, and 3.5 μL sterile dH2O. To the undigested tube was added 10 μL undigested master mix, and 4.5 μL sterile dH2O.

To prepare the unknown plasmid digest, 6.1 μL 41.1 ng/μL of isolated non-recombinant plasmid DNA was pipetted into three micro centrifuge tubes. To the double digest tube was added 12 μL double digest master mix, and 1.9 μL sterile dH2O. To the single digest tube was added 11 μL single digest master mix, and 2.9 μL sterile dH2O. To the undigested tube was added 10 μL undigested master mix, and 3.9 μL sterile dH2O.

Digests were then incubated at 37 °C for one hour and then stored at -20 °C for one week. After which the digestions were examined by gel electrophoresis. The samples were run on a 50 mL 0.9% (w/v) agarose gel in 1X TAE buffer at 100 V until the leading track dye traveled 2/3 the distance of the gel. The gel was then soaked in GelRed for 20 minutes and examined under UV light. To prepare the digestions 10 μL of each digestion was mixed with 2 μL of 6X track dye in a micro centrifuge tube. 12 μL of 1 kb DNA ladder and each digestion was run on the