Reference no: EM132994066

BC2023 Molecular Genetics - James Cook University

Manipulation of Plasmid DNA

Aim:
This purpose of this experiment was to investigate the effects of the restriction enzyme, HindIII, during the process of plasmid DNA digestion. HindIII was utilized at various concentrations (10x and undiluted) in order to clearly identify its impact. Arctic shrimp phosphatase and T4 DNA ligase were the enzymes implemented in order to examine the influence they have on plasmid DNA migration. Furthermore, a 1% agarose gel was prepared to run the various concentrations to characterise an unknown plasmid.

Methods and Materials:
Enzymatic reactions and 1% agarose gel were performed, and samples run as per the BC2023 laboratory manual with the following modifications:

• Tubes 1, 4 and 5 from the laboratory manual were combined into one tube due to them all containing the same concentrations for the initial 30-minute incubation period. Afterwards, the 60μL in the original microfuge tube was equally divided by pipetting 20μL into two new tubes, creating tubes 1, 4 and 5 as stated in the practical workbook.
• Tube 3 was disregarded, therefore, no 100x concentration was performed
• 30mL of 1% agarose gel was prepared instead of previously mentioned 40mL in laboratory manual
• 12μL from tube 1 (undiluted), tube 2 (10x concentration) and tube 6 (control - 0μL of
restriction enzyme) were loaded into the 1% agarose gel
• Tube 4 contained 1μL ligase plus 2.3μL ligase buffer. Tube 5 contained 1μL phosphatase, 2.3μL phosphatase buffer, 1μL ligase and 2.7μL ligase buffer
• Tubes 4 and 5 were incubated at 80°C for 10 minutes instead of 15 minutes as specified in the practical manual in order to inactivate the restriction enzyme
• Tubes 5 was incubated at 80°C for 10 minutes instead of 65°C for 15 minutes to
inactivate the phosphatase
• Only 1 DNA ladder was added between two groups due to its unavailability
• The following volumes were implemented in the restriction digest:
o 7μL - ddH2O (Tubes 1, 2, 4 and 5)
o 10μL - ddH2O (Tube 6 - control)
o 2μL - 10x restriction buffer
o 10μL - isolated plasmid DNA

Discussion and Conclusion:

This investigation aimed to identify the digestion of plasmid DNA whist utilizing the restriction enzyme, HindIII. Moreover, reagents such as arctic shrimp phosphatase and T4 DNA ligase were added to certain samples to observe the impact they have on the migration patterns of plasmid DNA. Figure 1 visually demonstrates that DNA migration of each sample in comparison to a 1KB DNA ladder. Tube 1 which contained the undiluted sample of HindIII, produced a singular band which migrated to approximately 3500 base pairs as noted in Table

1. The linear nature of this sample is represented by the minimal pattern of migration of this band as a result of the friction it sustains with the medium from it being less compact. Similarly, tube 2 which contained a 10x dilution of HindIII also displayed its heaviest band in roughly 3500 base pairs suggesting that it is linear due to the friction created with the 1% agarose gel during migration. This finding was also expected as the sample was diluted, therefore, there should not be significant migration. Sample 4 seen in Figure 1 showed bands at approximately 3500 base pairs which were thought to be nicked and linear in addition to 1500 base pairs which was presumed to be of supercoiled nature. Tube 4 contained undiluted HindIII plus 2.3μL of the reagent T4 DNA ligase which is responsible for creating the final phosphodiester bond to fully repair DNA. The mechanism of ligation is to create phosphodiester bonds between the 3' hydroxyl end of a nucleotide with the 5' phosphate end of another. The presence of this reagent is seen by the numerous small bands in the nicked section of Figure 1 ranging from 10000 to 3500 base pairs. This unexpected trend indicates that there was not enough of the ligase buffer to handle the increased quantity of DNA. It was expected that the ligase would have ligated the cut DNA, preventing the production of numerous bands in the nicked section, furthermore, a relegated circular plasmid band was sought after. Tube 5 contained undiluted HindIII restriction enzyme with the addition of 2.3μL of arctic shrimp phosphatase reagent plus 2.7μL of T4 DNA ligase. The primary function of phosphatase is to remove the phosphate groups in the 5' and 3' ends of nucleotides to prevent the vector from religating and in the process, enhances the cloning procedure. Table 1 exhibits that the band from sample 5 contained approximately 4000 base pairs and therefore, can be considered linear. As a result of the reagents present in this sample, it was expected that the band be linear as the cut DNA was not able to be ligated. Due to the absence of HindIII, the control sample run in tube 6 displayed an estimated 1500 base pairs, as specified in Table 1. It was expected that the band would be supercoiled as a result of the negligible amount of friction with the medium, allowing the sample to migrate further.

This practical was conducted in accordance with the BC2023 practical manual, however, errors may have occurred whilst measuring or pipetting the quantities of reagents, dye and/or buffer. Additionally, the pipettes which were used may not have been calibrated properly or the heating block may not have heated the DNA samples to the appropriate temperature due to the age of the equipment used. Contamination may also have been a factor in producing unexpected results from this experiment.

Attachment:- Molecular Genetics.rar