Miniplasmid DNA Preparation - Lab Report Example

Practical: Miniplasmid DNA preparation Introduction Plasmids are double DNA molecules that contain a wide variety of genes coding sequence. Some of the codes are for antibiotic restriction and resistance enzymes. For this reason, over-use of antibiotics can select the bacteria that contain plasmids and may amplify them to resist antibiotics. Plasmids can also be used to propagate foreign genes and are referred to as vectors (Casali & Preston, 2003). In this practical, purified plasmid pBR322 was isolated from E-coli strains grown in the presence of ampicillin. It consists of 4,361 base pairs together with tetracycline (Tet) and an antibiotic resistance genes for ampicillin (Amp). Ampicillin inhibits the growth of bacteria the interference cell wall synthesis. Its resistance leads to the production of betalactamase enzymes which are secreted by altered bacterial cells and can destroy the ampicillin around the agar medium. Aims: The aims for this practical were to: 1. Learn and get experience in pouring, setting up and running agarose gel electrophoresis. 2. Learn and get experience in isolating and purifying plasmid DNA from a bacterial cell culture. 3. Learn how to determine the size of DNA from a gel using molecular weight markers. 4. Learn a simple method of quantitating DNA and assessing its purity. Methods: Part A: Preparation of agarose gel The practical began with placing 0.5g of agarose into a beaker before adding 50mL of 1 x TAE buffer. Using the heat-proof gloves, the flask was heated carefully in the microwave, stopping occasionally to swirl the mixture to ensure proper melting. When the solution was completely clear, the flask was left at room temperature to cool for three minutes. Then 60° C water bath was poured to maintain that temperature. The gel tray ends were held firmly with a masking tape to from fluid-tight seal, before leveling on the workbench. The comb was inserted into the tray, making sure that it was closed to one end of the gel, and that the teeth of the comb were not touching the base of the tray. Finally, using the disposable gloves, 5μL of GelRed Nucleic Acid Stain (Botium, Inc) was added to the agarose at 60°C before pouring it into the tray while making sure that it did not dislodge the comb in the process. Any bubbles which appeared in the surface of the gel, was pricked with a needle before leaving the gel to set for 15-20 minutes. Part B: Isolation and Purification of Plasmid DNA This part began with putting the Binding Buffer on ice. The starting material was prepared by centrifuging the bacterial suspension at 10, 000 x g for 5 mins to forming bacterial cells of 0.5-4.0 ml of E. coli sample. The supernatant was discarded afterwards, before adding 250μl of Suspension Buffer containing RNase. The bacterial pellet was also resuspendend and mixed with vortexing and treated by adding 250 μl Lysis Buffer. They were then gently mixed by turning it upside down for about 5 times before incubating it for 5 minutes at 15 to 25°C. The lysed solution was also treated by adding 350 μl Binding Buffer and mixed gently before incubating on ice for 5 minutes prior to centrifuging it for 10 min at approx. 13,000 x g (full speed) in a microcentrifuge tabletop. DNA sample was first washed by adding 700 μl Wash Buffer II to the Filter Tube before centrifuging for 30 to 60 seconds at full speed. The assembly of the High Pure Tube was then centrifuge for an additional 30 to 60s to eliminate Wash Buffer residue. Then the Collection Tube was discarded and DNA was eluted by inserting a Filter Tube into a sterile, clean 1.5 ml microcentrifuge tube and adding 100μl Elution Buffer to the Filter Tube. Finally, the tube mixture was centrifuged for 30 to 60 seconds at full speed (13, 000 xg) Part C: Preparation of agarose gel and samples for sample loading After the gel was set, the comb was pulled out gently upwards until the seal between the gel and the comb was broken. After observing one end of the gel, the tape was removed from the gel tray edges and the tray placed into the gel tank; with the wells at the black electrode (cathode) end like as shown in the diagram below. Figure 1: DNA gel electrophoresis showing DNA as it run from the cathode to the anode 1 x TAE buffer was poured gently into the tank, taking care not to slide the gel off the tray before filling it until the gel was completely covered, with 1 – 2 mm of buffer above it. The samples were then loaded through the buffer into the wells. After noting the colour of the eluted plasmid DNA, 5µl of the DNA was added into a fresh eppendorf tube and labeled D. 5µl of ddH2O was also added to another tube labeled P, but 2µl of the loading dye was added to both tubes. They were then mixed thoroughly in each tube and the samples placed in the gel wells for electrophoresis. The solution was later dispersed over the eppendorf which was brought to the bottom in order to collect it into the loading pipette tip. The DNA molecular weight marker labeled M was obtained from the demonstrator. All the samples were heated at 65°C for 5 min and afterward placed on ice to enable DNA bands separation. Part D: Loading and running the gel In this section, 5µl of Pipette of the practice solution (P) was put in a pipette and loaded carefully into the first leftmost well in agarose gel. After changing the pipette tip the molecular weight marker (M) was loaded into the next leftmost well. The tip of the pipette was also changed before loading the DNA sample (D) into the next well. Then the chamber cover was slide put in place, and the power supply connected, making sure that anode and cathode were connected properly. Electrophorese at was done at 100 V for 1.5 hours. A sketch was drawn in laboratory records book of the gel and its lanes and the lane which had the M. W. markers and that which had samples were marked. The lanes were labeled for identification purpose. At the end of electrophoresis, the power supply was turned off and the leads disconnected from the power supply. After wearing double gloves, the gel tray from the chamber was removed and carefully placed in a tray. A photo of the gel was taken using the Chemidoc digital imaging system. Part E: Quantification of Plasmid DNA Using a Spectrophotometer This began with turning on the machine so as to warm up and then making sure that 2 clean cuvettes are available. The spectrophotometer was set to 260 nm. After diluting and measuring the samples, 1 mL of ddH2O was added into one of the cuvettes, before inserting it into the cuvette holder of the spectrophotometer. 5 µl of DNA of unknown concentration put into a clean matching cuvette, 1ml of water added and mixed by covering with parafilm and being inverted. The cuvette was inserted into machine, the reading noted and the value recorded for later analysis. The wavelength was reset to 280 before diluting and measuring other samples. Results: Table 1: Calculations and recording of molecular weight and distance moved by dye, molecular weight markers, plasmid and their Rf values Molecular weight (bp) Distance moved (cm) Rf (distance moved by sample cm/distance moved by dye cm) Dye (yellow) 5.75 1 Molecular weight markers 21226 1.2 0.21 5148 1.7 0.30 4973 1.8 0.31 4268 2.3 0.40 3530 2.5 0.43 2027 2.6 0.45 1904 3 0.52 1584 3.2 0.56 1375 3.6 0.63 947 831 564 Plasmid 3900 2.2 0.38 Table 1: Quantification of Plasmid DNA Using a Spectrophotometer Volume of plasmid DNA 1ml Volume (H2O) 1ml Total Volume 2ml OD 260 0.589AU OD280 0.646AU The amount of plasmid DNA isolated 2. The gel photo Figure 2: The gel photo 3. Gel electrophoresis photograph DNA marker: Figure 3: Gel electrophoresis photo (taken by chemidol digital imaging system) showing 2 groups results (Molecular weight markers and plasmid. 4. Distance moved from the well by the faster moving Blue Dye (Estimated size -500bp)=5.75 cm (1) (2) (3) (4) (5) (6). (7). (8). (9) Table2: Rf value of standard DNA Marker and the Plasmid band Molecular weight of the markers(in bp) Distance travelled by DNA bands(in cm) Rf Values 21226 1.2 5148 1.7 0.21 4973 1.8 0.30 4268 2.3 0.31 3530 2.5 0.40 2027 2.6 0.43 1904 3 0.45 1584 3.2 0.52 1375 3.6 0.56 947 0.63 831 564 Test Sample (Plasmid band): Molecular weight read off from the graph = 3900 2.2 0.38 The molecular weight of the isolated pBR322 was determined to be 3900 base pairs. A graph: Figure 4: Standard curve of relative distance migrated (Rf) versus molecular weight (number of base pair) Key: 0.38 and 3,900 is the relative distance migrated by isolated plasmid DNA and its molecular weight (bp) respectively. The amount of plasmid DNA isolated can be obtained from the following equation: Discussion Although only one plasmid has been isolated, there was more than one band on the gel. This is because plasmid has more than one DNA molecule. The molecules migrate as bands that are separated from each other in the lane of their migration. The rate of migration of the DNA molecule through the gel depends on it shape, size, the electrophoresis buffer type, voltage applied and the concentration of the gel. Supercoiled DNA has the fastest rate of migration different plasmid forms because it is more compact and lighter. Due to its supercoiled conformation, their sizes are small making it faster during migration because of less friction from the gel. Degraded RNA migrate faster than supercoiled DNA because of its small size (Casali & Preston, 2003). Gel photograph Plasmid DNA shows the wells for the mixtures of DNA fragments, but the gel electrophoresis photograph DNA markers shows the DNA bands formed by after DNA migration. Alkaline lysis method was used to isolate and purify pBR322 from E. coli cells since it is simple and faster method. The following steps were required in the purification of plasmids. a) A solution containing sodium dodecyl sulphate (SDS) is added to the suspended bacterial cells so as to make plasma membrane porous and denature cellular proteins. It also facilitate in the DNA purification process. b) The solution is made very alkaline because: NaOH denature DNA which maintains the cell membrane structure. It does this by breaking H-bonds between complementary base pairs; making the double-stranded chromosomal and plasmid DNA single-stranded. Through removal of supernatant and agitation, the cellular is removed and the plasmid is isolated, thus also aiding the purification process. c) The purpose of potassium acetate buffer is for neutralisation and therefore reduces the alkalinity of the solution. This enables the small plasmid DNA to re-nature. d) Isopropanol is added in the cleaning step (wash) to precipitate DNA out of solution. In the conventional purification performed in the practical, filtration was utilised, and the use of isopropanol was not necessary. e) The pellet is suspended in Tris buffer which contains EDTA. The purpose of using EDTA in re-suspension was to chelate divalent cations in solution (e.g., Ca²⁺ and Mg²⁺), which are required for the activity of DNase, and thus prevent DNase from damaging plasmid (Casali & Preston, 2003). f) RNase (ribonuclease) was added so as to degrade cellular RNA. Conclusions The amount of plasmid DNA isolated was 42.9μg/ml. However, the ratio of absorbance at 260nm to 280nm was 1.3 being out of 1.8 - 2.0 range. Since the value is below the range, it indicates that the isolated plasmid DNA was not pure and was contaminated with protein. The most likely reason for this impurity could be insufficient cleaning during purification, which led to some residual cellular protein being eluted with the plasmid DNA. A single plasmid band was visible as in figure 3 above. This indicates that the isolated pBR322 plasmids were all in the same form (most likely super-coiled as it is the common form). As it can be seen from figure 4 the molecular weight of the isolated pBR322 was determined to be 3900 base pairs (Calladine, 2004). The information learnt from this experiment includes: - How to set up and run agarose gel electrophoresis analysis on my own. - Practical experience in doing experiment especially in this case isolating and purifying plasmid DNA from a sample. - How to determine the size of DNA from a gel using molecular weight markers. - A simple and practical way of quantitating DNA and assessing its purity. I have through practical experience how to isolate plasmid, which will assist in future research work. In future work I would try to eliminate error so as to obtain more accurate results. References: Calladine, C. R., 2004. Understanding DNA the molecule & how it works. San Diego, CA, Elsevier Academic Press. http://www.123library.org/book_details/?id=44360. Casali, N., & Preston, A., 2003. E. coli plasmid vectors: methods and applications. Totowa, N.J., Humana Press. Giri, C. C., & Giri, A., 2007. Plant biotechnology: practical manual. New Delhi [u.a.], I.K. International Publ. House. Helgason, C. D., & Miller, C. L., 2013. Basic cell culture protocols. New York, Humana Press. Reece, R. J., 2004. Analysis of Genes and Genomes. Chichester, John Wiley & Sons. http://www.123library.org/book_details/?id=15034 S. Harisha, & S. Harisha, 2006. Introduction to practical biotechnology: a hand book on practical biotechnology. Bangalore, Laxmi Publication. Micklos, D. A., & Freyer, G. A., 1990. DNA science: a first course in recombinant DNA technology. Burlington, N.C., Carolina Biological Supply Co. Krebs, J. E., Lewin, B., Goldstein, E. S., Kilpatrick, S. T., & Lewin, B., 2013. Lewin's essential genes. Burlington, MA, Jones and Bartlett Learning. Robert A. Welch Foundation., 1985. Genetic chemistry: the molecular basis of heredity : November 4-6, 1985, the Shamrock Hilton Hotel, Houston, Texas. Houston, Tex, Robert A. Welch Foundation. Palmer, T., & Bonner, P. L., 2007. Enzymes biochemistry, biotechnology and clinical chemistry. Oxford, Woodhead Publishing. Appendix: quantification of concentration of purity plasmid Read More

DNA sample was first washed by adding 700 μl Wash Buffer II to the Filter Tube before centrifuging for 30 to 60 seconds at full speed. The assembly of the High Pure Tube was then centrifuge for an additional 30 to 60s to eliminate Wash Buffer residue. Then the Collection Tube was discarded and DNA was eluted by inserting a Filter Tube into a sterile, clean 1.5 ml microcentrifuge tube and adding 100μl Elution Buffer to the Filter Tube. Finally, the tube mixture was centrifuged for 30 to 60 seconds at full speed (13, 000 xg) Part C: Preparation of agarose gel and samples for sample loading After the gel was set, the comb was pulled out gently upwards until the seal between the gel and the comb was broken.

After observing one end of the gel, the tape was removed from the gel tray edges and the tray placed into the gel tank; with the wells at the black electrode (cathode) end like as shown in the diagram below. Figure 1: DNA gel electrophoresis showing DNA as it run from the cathode to the anode 1 x TAE buffer was poured gently into the tank, taking care not to slide the gel off the tray before filling it until the gel was completely covered, with 1 – 2 mm of buffer above it. The samples were then loaded through the buffer into the wells.

After noting the colour of the eluted plasmid DNA, 5µl of the DNA was added into a fresh eppendorf tube and labeled D. 5µl of ddH2O was also added to another tube labeled P, but 2µl of the loading dye was added to both tubes. They were then mixed thoroughly in each tube and the samples placed in the gel wells for electrophoresis. The solution was later dispersed over the eppendorf which was brought to the bottom in order to collect it into the loading pipette tip. The DNA molecular weight marker labeled M was obtained from the demonstrator.

All the samples were heated at 65°C for 5 min and afterward placed on ice to enable DNA bands separation. Part D: Loading and running the gel In this section, 5µl of Pipette of the practice solution (P) was put in a pipette and loaded carefully into the first leftmost well in agarose gel. After changing the pipette tip the molecular weight marker (M) was loaded into the next leftmost well. The tip of the pipette was also changed before loading the DNA sample (D) into the next well. Then the chamber cover was slide put in place, and the power supply connected, making sure that anode and cathode were connected properly.

Electrophorese at was done at 100 V for 1.5 hours. A sketch was drawn in laboratory records book of the gel and its lanes and the lane which had the M. W. markers and that which had samples were marked. The lanes were labeled for identification purpose. At the end of electrophoresis, the power supply was turned off and the leads disconnected from the power supply. After wearing double gloves, the gel tray from the chamber was removed and carefully placed in a tray. A photo of the gel was taken using the Chemidoc digital imaging system.

Part E: Quantification of Plasmid DNA Using a Spectrophotometer This began with turning on the machine so as to warm up and then making sure that 2 clean cuvettes are available. The spectrophotometer was set to 260 nm. After diluting and measuring the samples, 1 mL of ddH2O was added into one of the cuvettes, before inserting it into the cuvette holder of the spectrophotometer. 5 µl of DNA of unknown concentration put into a clean matching cuvette, 1ml of water added and mixed by covering with parafilm and being inverted.

The cuvette was inserted into machine, the reading noted and the value recorded for later analysis. The wavelength was reset to 280 before diluting and measuring other samples. Results: Table 1: Calculations and recording of molecular weight and distance moved by dye, molecular weight markers, plasmid and their Rf values Molecular weight (bp) Distance moved (cm) Rf (distance moved by sample cm/distance moved by dye cm) Dye (yellow) 5.75 1 Molecular weight markers 21226 1.2 0.

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