Ligation of the EGFP insert to the plasmid vector Ligation of the EGFP cDNA into a pET41a(+) plasmid to create recombinant expression plasmids and perform these ligations through gel electrophoresis to visualize the DNA and verify the success of the ligations. Five ligation reactions were generated, two actual ligations and three controls, with a total final volume of 20 µL each. NcoI and NotI are restriction endonucleases whose purpose is to reduce the formation of non-recombinant plasmids and prevent unwanted rearrangements during ligation. The first ligation was a 1:1 molar ratio pET-41a(+) vector:egfp insert using 50ng NotI/NcoI shear pET-41a(+) DNA, 7ng egfp insert DNA, 1uL DNA ligase and the appropriate amount of water to dilute the ligase buffer 10x to a final concentration 1x. Ligation two was a 1:3 molar ratio pET-41a (+) vector: egfp insert composed of 50 ng of NcoI/NotI (+) cleaved pET-41a, 21 ng of egfp insert DNA, 1 uL of DNA ligase and just the right amount of water to dilute the ligase buffer 10x to a final concentration 1x. The water has been sterilized and deionized. The remaining three ligation samples served as controls. Ligation three contained 57 ng of uncut pET-41a(+)/EGFP recombinant plasmid DNA and sterile water. Ligation 4 was a negative control consisting of sterile water only. Ligation five is devoid of DNA ligase but has the same properties as the pET-41a(+)/EGFP vector at a 1:3 molar ratio. The supplied DNA ladder sample and each individual ligation sample were analyzed on 40 ml of 0.8% agarose in 1x TAE buffer for approximately sixty minutes at 110V. The appropriate volume of 6x GelRed dye was used after being diluted to a final concentration of 1x and incubated for thirty minutes. Finally, the gel was illuminated under UV light and analyzed. Between...... middle of the paper...... Gave access to virtually annotate sequences freely, build and visualize maps, design primers, and restriction analyses. First, the nucleotide sequence of plasmid pEGFP-N1 was found using the NCBI nucleotide database program. The SnapGene viewer illustrated the restriction enzyme cleavage sites used to cut the EGFP gene from the source plasmid pEGFP-N1. Then the pET-41a(+) vector sequence was found using the AddGene vector database. A new DNA file representing the recombinant pET-41a(+)-EGFP plasmid was created by virtually cloning the EGFP gene insert into the pET-41a(+) vector sequence. The plasmid was virtually cut using sense primer pAD1 and anti primer pAD1 by the PCR procedure. A restriction digestion experiment was designed to confirm the identity of the PCR product. The two restriction endonucleases that cleave the PCR product at least once were HgaI and XspI.