MED3LAB SLT Presentation

WhatsApp Video 2021-10-25 at 7.33.19 PM.mp4. MED3LAB SLT Presentation.

Ligation of vector and insert.

WhatsApp Video 2021-10-25 at 7.34.03 PM.mp4. Qn 1: Within the practical module, we will set up some ligations. What is the endogenous function of DNA ligase?.

WhatsApp Video 2021-10-25 at 7.34.33 PM.mp4. Qn 1: How about T4 DNA ligase in particular?.

Slide 5.mov. Qn 2: Why is it often important in ligation reactions to optimise the molar ratio of insert to vector and also the overall DNA concentration?.

Slide 6(edited).mp4. Qn 2: Why is it often important in ligation reactions to optimise the molar ratio of insert to vector and also the overall DNA concentration?.

WhatsApp Video 2021-10-23 at 7.07.09 PM.mp4. Qn 3: Some ligation reaction protocols suggest incubating for a short time at room temperature while others suggest a longer incubation at about 14°C. Why might researchers sometimes use the lower temperature?.

Transformation into E. coli cells.

WhatsApp Video 2021-10-23 at 7.49.05 PM.mp4. Qn 1: What is a “competent” cell? When preparing the competent cells by the Inoue method, why do we grow the bacteria at 18°C overnight?.

WhatsApp Video 2021-10-25 at 1.14.25 PM.mp4. Qn 2: In this practical module, the competent cells are transformed via heat shock. How does this method work? Describe another method to achieve artificial transformation of bacteria..

WhatsApp Video 2021-10-23 at 7.07.14 PM.mp4. Qn 3: How could we calculate the transformation efficiency of competent cells?.

References. Anders H. Lund, Mogens Duch, Finn Skou Pedersen, Increased Cloning Efficiency by Temperature-Cycle Ligation, Nucleic Acids Research, Volume 24, Issue 4, 1 February 1996, Pages 800–801, https://doi.org/10.1093/nar/24.4.800 C, B. G. (2021, February 23). Bacterial Transformation – The Science Notes . The Science Notes. Retrieved October 21, 2021, from https://www.thesciencenotes.com/bacterial-transformation/ Carter, M., & Shieh, J. (2015). Gene Delivery Strategies. Guide to Research Techniques in Neuroscience, 239–252. https://doi.org/10.1016/b978-0-12-800511-8.00011-3 Cotner-Gohara, E., Kim, I. K., Hammel, M., Tainer, J. A., Tomkinson, A. E., & Ellenberger, T. (2010). Human DNA ligase III recognizes DNA ends by dynamic switching between two DNA-bound states. Biochemistry , 49 (29), 6165–6176. https://doi.org/10.1021/bi100503w Crow, E. (2010, December 15). Ligation optimization . Bitesize Bio. Retrieved October 21, 2021, from https://bitesizebio.com/10203/ligation-optimization/ Kroemer, T. (n.d.). Overview of T4 DNA Ligase: What it is, how it works, reactions, and more | GoldBio . Goldbio. Retrieved October 21, 2021, from https://www.goldbio.com/articles/article/overview-of-T4-DNA-ligase Lund, A. H., Duch, M., & Skou Pedersen, F. (1996). Increased Cloning Efficiency by Temperature-Cycle Ligation. Nucleic Acids Research , 24 (4), 800–801. https://doi.org/10.1093/nar/24.4.800 Martin, I. V., & MacNeill, S. A. (2002). ATP-dependent DNA ligases. Genome Biology , 3 (4), reviews3005.1. https://doi.org/10.1186/gb-2002-3-4-reviews3005 Rahimzadeh, M., Sadeghizadeh, M., Najafi, F., Arab, S., & Mobasheri, H. (2016). Impact of heat shock step on bacterial transformation efficiency. Molecular biology research communications, 5(4), 257–261. Tomkinson, A. E., & Sallmyr, A. (2013). Structure and function of the DNA ligases encoded by the mammalian LIG3 gene. Gene , 531 (2), 150–157. https://doi.org/10.1016/j.gene.2013.08.061 Zhou, J., Li, X., Xia, J., Wen, Y., Zhou, J., Yu, Z., & Tian, B. (2018). The role of temperature and bivalent ions in preparing competent Escherichia coli . 3 Biotech , 8 (5), 222. https://doi.org/10.1007/s13205-018-1243-x.

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