Lab report
LSM1102 Lab Report
Introduction
Transformation is a process which involves plasmid DNA being bound to the cell surface and the subsequent uptake of DNA by the cell (Panja et al., 2008). For artificial transformation of E. coli cells with plasmids, plasmid DNA has to be extracted from bacterial cells using the High-Speed Plasmid Mini Kit, which is then mixed with competent E. coli cells followed by heat shock and the streaking of transformed cells on two different types of agar plate (LB and LB+ampicillin). The extracted plasmid DNA is important as it contains ampicillin-resistant gene. As such, E. coli cells that have taken up this plasmid DNA will be resistant to ampicillin and survive, hence growth of colonies will be observed on the agar plates. One of the rationales behind heat shock method is to create pores, allowing uptake of plasmid DNA (Panja et al., 2008). For this practical, another ice incubation step is added in after heat shock at 42ºC to investigate its significance in increasing the efficiency of transformation. It is hypothesized that the additional ice incubation step after heat shock will cause more DNA plasmids uptake by competent E. coli cells and thus more growth of colonies of successful transformants, resulting in the increase of transformation efficiency. By comparing the results of both standard and mutated protocols, the hypothesis on the effect of post heat shock ice incubation step on transformation efficiency can then be known to be true or false.
Materials
1. E. coli (pUC18) culture
2. Geneaid Spin Column with glass fiber matrix
3. Microcentrifuge tubes
4. Buffer PD1 (50 mM Tris-HCl pH 8.0; 10 mM EDTA; 10 μg/ml RnaseA)
5. Buffer PD2 (200 mM NaOH; 1% SDS (w/v))
6. Buffer PD3 (guanidinium hydrochloride and acetic acid)
7. W1 Buffer (guanidine hydrochloride and isopropanol)
8. Wash Buffer (70% ethanol)
9. Elution Buffer (10 mM Tris-HCl, pH8.5 at 25°C)
10. 1 ml of competent E. coli cells in 100mM CaCl2
11.
Introduction
Transformation is a process which involves plasmid DNA being bound to the cell surface and the subsequent uptake of DNA by the cell (Panja et al., 2008). For artificial transformation of E. coli cells with plasmids, plasmid DNA has to be extracted from bacterial cells using the High-Speed Plasmid Mini Kit, which is then mixed with competent E. coli cells followed by heat shock and the streaking of transformed cells on two different types of agar plate (LB and LB+ampicillin). The extracted plasmid DNA is important as it contains ampicillin-resistant gene. As such, E. coli cells that have taken up this plasmid DNA will be resistant to ampicillin and survive, hence growth of colonies will be observed on the agar plates. One of the rationales behind heat shock method is to create pores, allowing uptake of plasmid DNA (Panja et al., 2008). For this practical, another ice incubation step is added in after heat shock at 42ºC to investigate its significance in increasing the efficiency of transformation. It is hypothesized that the additional ice incubation step after heat shock will cause more DNA plasmids uptake by competent E. coli cells and thus more growth of colonies of successful transformants, resulting in the increase of transformation efficiency. By comparing the results of both standard and mutated protocols, the hypothesis on the effect of post heat shock ice incubation step on transformation efficiency can then be known to be true or false.
Materials
1. E. coli (pUC18) culture
2. Geneaid Spin Column with glass fiber matrix
3. Microcentrifuge tubes
4. Buffer PD1 (50 mM Tris-HCl pH 8.0; 10 mM EDTA; 10 μg/ml RnaseA)
5. Buffer PD2 (200 mM NaOH; 1% SDS (w/v))
6. Buffer PD3 (guanidinium hydrochloride and acetic acid)
7. W1 Buffer (guanidine hydrochloride and isopropanol)
8. Wash Buffer (70% ethanol)
9. Elution Buffer (10 mM Tris-HCl, pH8.5 at 25°C)
10. 1 ml of competent E. coli cells in 100mM CaCl2
11.
References: Bergmans, H. E., Van Die, I. M., & Hoekstra, W. P. (1981). Transformation in Escherichia coli: Stages in the Process Panja, S., Aich, P., Jana, B., & Basu, T. (2008). How does plasmid DNA penetrate cell membranes in artificial transformation process of Escherichia coli? Molecular Singh, M., Yadav, A., Ma, X., & Amoah, E. (2010). Plasmid DNA Transformation in Escherichia coli: Effect of Heat Shock Temperature, Duration, and Cold Incubation Wilfinger, W. W., Mackey, K., & Chomczynski, P. (1997). Effect of pH and Ionic Strength on the Spectrophtometric Assessment of Nucleic Acid Purity