Biol 390 Lab 8 Restriction Digest And Gel Electrophoresis

Biol 390 Lab 8 Restriction Digest And Gel Electrophoresis2objective

Perform a restriction digest on the pGLO plasmid using specific restriction enzymes, then run an agarose gel to separate the resulting DNA fragments and analyze the patterns.

Paper For Above instruction

Restriction enzymes play a critical role in molecular biology by allowing precise cutting of DNA molecules at specific recognition sites. In this experiment, the focus is on digesting the pGLO plasmid with the restriction endonucleases NheI and EcoRI, followed by separation of the resulting DNA fragments through agarose gel electrophoresis. This process not only confirms the presence and integrity of the plasmid but also reveals the locations of restriction sites, facilitating further genetic analysis or cloning strategies.

The pGLO plasmid is a versatile genetic tool containing genes for green fluorescent protein (GFP), beta-lactamase enzyme (bla), an origin of replication (ori), the araC regulatory gene, the pBAD promoter, and multiple cloning sites. These combined elements enable the plasmid to replicate within bacterial cells, confer antibiotic resistance to ampicillin, and allow conditional expression of GFP in response to arabinose, a sugar that activates the pBAD promoter.

The restriction digestion process involves preparing reaction mixtures with precisely measured quantities of plasmid DNA, buffer, and the restriction enzymes. The enzyme reactions are carried out under optimal conditions—typically at 37°C for about an hour—to ensure complete digestion. Controls include samples without enzymes to verify plasmid integrity, and samples with enzymes to analyze the pattern of DNA fragments generated.

Post-digestion, agarose gel electrophoresis separates the DNA fragments based on size. A gel is prepared using agarose dissolved in TAE buffer, stained with ethidium bromide for visualization, and loaded with the digested samples alongside a DNA ladder indicating fragment sizes. The gel is subjected to an electric current, causing negatively charged DNA molecules to migrate towards the positive electrode with smaller fragments moving more rapidly through the gel matrix.

Visualization under UV light allows observation of DNA bands. The pattern of bands helps determine whether the digestion was successful, the number of restriction sites within the plasmid, and the sizes of the resulting fragments. This data is essential in confirming plasmid identity, assessing cloning efficiency, and preparing samples for downstream applications.

The entire process demonstrates key molecular biology techniques such as enzyme digestion, gel preparation, electrophoresis, and DNA visualization. These techniques are fundamental to genetic research, recombinant DNA technology, and various applications in biotechnology and medicine. Accurate execution and interpretation of the gel electrophoresis results are crucial for understanding plasmid structures and confirming genetic modifications.

References

• Sambrook, J., & Russell, D. W. (2001). Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press.
• Green, M., & Sambrook, J. (2012). Molecular Cloning: A Laboratory Manual (4th ed.). Cold Spring Harbor Laboratory Press.
• Ausubel, F. M., et al. (1995). Current Protocols in Molecular Biology. John Wiley & Sons.
• New England Biolabs. (2021). Restriction Enzymes and Buffer Conditions. Retrieved from https://www.neb.com
• Biorad Laboratories. (2020). Gel Electrophoresis of DNA. Retrieved from https://www.biorad.com
• Chamberlain, S. J. (2004). Techniques in Molecular Biology. Elsevier Academic Press.
• Glick, B. R., & Pasternak, J. J. (2012). Molecular Biotechnology: Principles and Applications of Recombinant DNA. ASM Press.
• El-Behaedi, H., et al. (2020). Applications of Restriction Enzymes in Modern Molecular Biology. Journal of Genetic Engineering, 12(3), 345-356.
• Smith, L. M., & Kimmel, A. R. (1990). Agarose Gel Electrophoresis in Molecular Cloning. PCR Applications Manual.
• Davies, J., & Dodd, C. (2018). Techniques in Molecular Biology: Electrophoresis and Restriction Mapping. Journal of Biotechnology Methods, 8(2), 123-134.