Lab Report 10

docx

School

Brigham Young University *

*We aren’t endorsed by this school

Course

384

Subject

Chemistry

Date

Jan 9, 2024

Type

docx

Pages

Uploaded by GeneralStarOctopus37

Karissa Williams CHEM 384, section 001 Dr. Ken Christensen 4/4/2023 Confirmation of Cloning Product In this lab we obtained evidence that we correctly cloned our pNCS-mNeonGreen plasmid. We inoculated several E. coli cultures from our colonies we produced in the previous lab with our cloning product of pNCS-mNeonGreen. We then obtained our confirmation of cloning through an analytical restriction digest reaction. An analytical digest reaction can be useful to verify a ligation reaction because it allows us to confirm the presence of the expected DNA fragment in the ligation product. In an analytical digest, the DNA is restriction digested, and resulting fragments are separated by gel electrophoresis and visualized to confirm the size and identity of the DNA fragments present. In the case of a ligation reaction, an analytical digest can be used to confirm that the DNA fragment of interest has been successfully incorporated into the plasmid vector. If the ligation reaction was successful, the expected DNA fragment should be present in the sample after digestion. In biochemical research, it is crucial to verify the success of a ligation to ensure accurate data reporting and experimentation with the correct DNA. To help verify successful ligation, we selected two colonies from our transformation plate and performed a miniprep on the colonies using the Zymo Plasmid Miniprep protocol. Then we purified the DNA we obtained with the clean & concentrate protocol and saved some of the product in order to run in our gel alongside the digest product. We then performed a restriction digest reaction with the miniprep product of the pNCS-mNeonGreen DNA, using the same enzymes BamHI and EcoRI. The digested plasmid was then loaded onto a gel alongside a sample of known mNeonGreen DNA obtained in lab 5. We also measured the resulting DNA from our Miniprep and cleanup on the NanoDrop to confirm DNA concentration. We then also decided to run an agarose gel after purifying our restriction digest product with clean & concentrate to separate the mNeonGreen DNA fragment and running it alongside a known fragment of mNeonGreen DNA to prove that the DNA fragment we obtained is mNeonGreen. Our results suggest that we successfully cloned of our pNCS-mNeonGreen plasmid. Figure 1A shows the agarose gel containing our restriction digest DNA product. The restriction digest products are run on our gel, resulting in the cut mNeonGreen and pNCS backbone being very visible in the third lane. The second lane shows the plasmid DNA we saved after the miniprep of our colonies before we ran the restriction digest so it is uncut. The third lane has a bright, distinct band that didn’t travel far indicating the larger pNCS backbone, and a fainter band that traveled farther indicating the smaller mNeonGreen DNA. In Figure 1B, The lane 3 of the processed plasmid displays a clearly visible band which migrated almost identically to the mNeonGreen

DNA of known length in lane 2, indicating the possibility of the colony's accurate clone. Table 1 shows the NanoDrop measurements of the purified plasmid DNA from our inoculated fluorescent colonies, which produced DNA at 155.4 ng/ul with acceptable purity ratios to continue forward. This data suggests that we successfully obtained at least one colony with the correct clone of our pNCS-mNeonGreen plasmid. Table 1. UV-Vis Verified pNCS-mNeonGreen Concentration and Statistics using the ThermoFisher NanoDrop. DNA Analyzed ng/ul A260/A280 A260/A230 Purified pDNA product 155.4 1.91 1.91 Figure 1. Agarose Gel Electrophoresis Product of Analytical Restriction Digest. A. UV image of verification of our analytical restriction digest product. Lane 1 is the 1kb DNA ladder. Lane 2 shows our plasmid DNA we obtained from our fluorescent colonies after we

miniprepped and purified the DNA. Lane 3 shows our restriction digest product, with obvious bands showing the pNCS backbone and the mNeonGreen fragment. B. UV image of cut mNeonGreen DNA further verifying that our DNA fragment produced in our restriction digest was in fact mNeonGreen. Lane 1 is the 1kb DNA ladder. Lane 2 contains our plasmid DNA, and lane 3 contains the known linear version of the mNeonGreen DNA. The bands produced are somewhat close together, showing that the correct DNA was produced in our cloning experiment.

Your preview ends here

Eager to read complete document? Join bartleby learn and gain access to the full version