Ligation Lab Report

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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
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Coli cells. Four transformations were prepared from ligations #1 - #4. 20 μL of E. Coli cell suspension was added to four 1.5 mL micro centrifuge tubes. Then to one tube each 2 μL of ligations #1 - #4 was added. Transformation #1 corresponds to the tube to which ligation #1 was added, and so on for each of the four transformations. After ligations were added each solution was chilled on ice for 5 minutes then heat shocked at 42° C for 5 minutes, then again chilled on ice for 2 minutes. To each tube was then added 80 μL room temperature Luria Broth. After which the solutions were incubated at 37° C for 45 minutes. Each solution was then plated on an LB/kanamycin/IPTG plate. Additionally transformation #2 was plated on a LB/kanamycin plate, and transformation #4 was plated on an LB plate. The purpose of including kanamycin on the plates was to selectively screen for plasmid containing E. Coli cells. The IPTG was to selectively screen for E. Coli cells with the plasmid containing the EGFP insert.

The plates were then incubated over night at 37° C.

DNA Purification
The objective of this experiment was to isolate plasmid DNA. Three bacterial cultures were received from laboratory staff. The
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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 gel.

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