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Abstract
An important method used in biology is plasmid purification. What makes this method so important is because a purified plasmid sample is essential for many experiments, including important techniques like DNA sequencing. Purified E.coli plasmid pBR322 using gel electrophoresis and a calibration curve were used in this experiment to quantify the size of the purified plasmid. Examples were examined using gel electrophoresis and were measured to the known size of pBR322, this helped determined if the plasmid was the target plasmid. The results showed that it was the target plasmid but the shape of the purified DNA was more compact, since the results showed a band much lower, 3113, than the standard marker for the literature value of 4361 base pairs.
Introduction
The purpose of this experiment was to purify the plasmid pBR322 of the bacteria strain Escherichia coli, these are important bacteria that can be found in human , they usually dont impose harm, but they can be pathogenic, infections etc. The main function of plasmid purification is to separate the plasmid DNA from cellular RNA and chromosomal DNA of the bacteria. A way to obtain this is through a technique which uses a traditional alkaline lysis method with a phenol/chloroform extraction and a subsequent precipitation using ethanol as a clean-up step. DNA then is analyzed through restriction digestion and run through a gel-electrophoresis. The use of gel electrophoresis is good for analyzing relative sizes of molecules. After applying an electric current, the negatively charged DNA backbone separates from the loading well terminal, that is negatively charged, to the positive end of the machine, by which it is attracted. The electrophoresis is run on a 1 % agarose gel, which contains pores through which the molecules will have to travel. Since DNA can have various shapes including supercoiled. Supercoiled DNA is partly unwound, it compacts DNA and is better in interacting with biomolecules. The shape of DNA is important when determining the distance of bands that travelled in the gel electrophoresis. If the DNA is more compact, then it travels further down.
Procedures
First 10 microliters of the plasmid were transferred with a P-20 micropipette into a new and clean 1.5 mL microfuge tube. Then, 2 microliters of 6X loading dye were added. Each student then transferred their sample into the well of the DNA ladder for the gel-electrophoresis. The gel was run at 100 Volts for 60 minutes and stained with protein pre-stain solution for another 50 minutes. 1.5 mL of E. coli cells were transferred and centrifuged for 2-3 minutes at 13,l000 rpm.. This process was repeated two times. The bacterial cells in the pellet were then put in 250 microliters of buffer P1. Then, 250 microliters of buffer P2 was added to the above solution and inverted. To this 350 microliters of buffer N3 were added, mixed and centrifuged for 10 minutes at 13,000 rpm. When centrifugation was complete, the supernatant was pipetted into a QIAprep spin column, centrifuged for 1 more minute and the flow-through was discarded. The QIAprep spin column was then washed with 0.75 mL of buffer PE, that contained 10 mM Tris-HCl and 80% ethanol and causes the plasmid DNA to stay on the column. The flow-through was again discarded and centrifuged for an additional 2 minutes to remove residues of wash buffer. The QIAprep column was then placed into a clean 1.5 microfuge tube and 50 microliters of buffer EB (10 mM Tris-HCl) were added to elute DNA. The mixture then left in the incubator for five minutes and centrifuged for 2 minutes. The final samples were stored.
Discussion
The results exhibited that the size of the purified plasmid pBR322 was 3113 base pairs, this indicates that is not close to the 4361 base pairs. Because of this, you can assume that the plasmid was more compact in shape, and as discussed before, the more compact in shape the further it travels down the gel. Gel electrophoresis separates molecules depending on their size, the results showed that the purified plasmid was not linear. Because if it was, it would be further up. The technique used for the purification of plasmid pBR322 was the Qiagen Mini prep-kit, this kit has all that was necessary in order to conduct the experiment. This method is good to use because it is easy and can be done in a few hours. Since the size in base pairs was less than the one expected, this shows that there were errors. Sources of error could have been wrong volumes of buffers, which does not give the ideal mixtures. Another possible error also could have been wrongly prepared agarose gel, this might have interfered or change the distance traveled.
References
- Ninfa, Alexander J. Fundamental Laboratory Approaches for Biochemistry and Biotechnology, 2nd Edition. 2. John Wiley & Sons, Inc, 2017
- Biochemistry(8th ed.).
- Berg, J. M., Tymoczko, J. L., Stryer, L., & Gumport, R. I. (2006).
- Basingstoke: W.H. Freeman & Co.
- Calibration Curve for Plamid DNA Purification from E.coli
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