BIOL10401 Online Practical 5: Plasmid DNA Purification PDF

11/23/23, 3:41 PM BIOL10401 Introduction to Laboratory Science BIOL10401 Introduction to Laboratory Science Online Practical 5: Plasmid DNA Purification and Analysis > Online Practical 5: Plasmid DNA Purification Dr Shazia Chaudhry Aims...

11/23/23, 3:41 PM BIOL10401 Introduction to Laboratory Science BIOL10401 Introduction to Laboratory Science Online Practical 5: Plasmid DNA Purification and Analysis > Online Practical 5: Plasmid DNA Purification Dr Shazia Chaudhry Aims and Outcomes You will learn how plasmid DNA can be isolated from bacterial cells and visualised by using agarose gel electrophoresis At the end of this online practical you will be able to: Transferable research skills Describe how DNA molecules are separated on the basis of size and visualised (agarose gel electrophoresis)​​ Explain how to separate small amounts of solid matter from liquid (bench-top microcentrifugation) Explain how to measure and deliver small volumes of liquids accurately (automatic and glass pipettes) Specific biological techniques Explain how to isolate plasmid DNA from bacterial cells Describe how to load, run and interpret an agarose gel containing nucleic acid Recognise the different conformations that plasmid DNA can adopt in agarose gels ​ Useful Data-handling Skills Modules 'Measurements and units' 'Moles and concentrations' 'Accuracy and precision' ​ ​ https://softchalkcloud.com/lesson/files/zxg6UA4wBGk1T8/Practical4_202021_print.html 1/13 11/23/23, 3:41 PM BIOL10401 Introduction to Laboratory Science Summary of Online Practical 5 Research in biology has been transformed in the last 40 years by the development of recombinant DNA technology which allows the molecular cloning of genes. This technology has enabled scientists to study the structure and function of genes in organisms ranging from bacteria to humans and has revolutionised our understanding of genes and how they function. The practical applications of the technology include the identification of disease-causing genes in humans and the production of human proteins (such as insulin and erythropoietin, EPO) in microorganisms and tissue culture cells for use as pharmaceuticals. Recombinant DNA technology was developed from basic research on prokaryotes, particularly the bacterium Escherichia coli (E. coli). This species is found in the intestines of warm-blooded mammals and is very easy to grow and manipulate in the laboratory. Some strains of E. coli are pathogenic for humans (such as E. coli O157) but the K12 strains used in teaching /research laboratories are harmless and have been used safely for many years. In Online Practical 5 you will learn how to perform an experiment using E. coli. Here we introduce the very common technique of purifying (isolating) plasmid DNA from E. coli and analysing the plasmid DNA using agarose gel electrophoresis. https://softchalkcloud.com/lesson/files/zxg6UA4wBGk1T8/Practical4_202021_print.html 2/13 11/23/23, 3:41 PM BIOL10401 Introduction to Laboratory Science What are plasmids? (background theory) Plasmids are extra-chromosomal pieces of DNA found naturally in many bacteria. They are usually circular, composed of double- stranded DNA, and can replicate inside bacterial cells alongside the chromosome. They are not essential for bacterial survival, but plasmid DNA often contains genes for one or more traits that may be beneficial to the host, e.g. genes for antibiotic resistance or for heavy metal metabolism. The genes encoded by the plasmid DNA can be expressed within the bacterial cell and can confer their functions upon the cell. Plasmids can move both from cell to cell ('horizontal' transmission) and to daughter cells following cell division ('vertical' transmission). Plasmids either replicate once per cell division - so that only one copy of the plasmid is found per cell (low copy number) - or they can replicate autonomously throughout the cell cycle of the bacteria, accumulating to hundreds of copies per cell (high copy number). They range in size from approximately 1000 DNA base-pairs up to several thousand kilobase-pairs. As well as occurring naturally, plasmids can be artificially engineered and used for many laboratory procedures such as DNA cloning or protein expression studies. As such, they are very useful tools for the molecular biologist. The plasmids in this practical, pBluescript and pGLO are two such cloning vectors. Plasmid DNA isolation Isolation of plasmid DNA from E. coli is a routine procedure performed in many research laboratories. The procedure, often referred to as a plasmid "mini-prep", involves the following key steps: 1. Harvesting a sufficient quantity of bacterial cells that harbour the plasmid 2. Breaking open the bacterial cells 3. Precipitating cellular debris (proteins, chromosomal DNA etc.) leaving the plasmid DNA in solution 4. Precipitating the plasmid DNA out of solution The procedure can be modified to include several 'clean-up' steps to improve the purity of the isolated DNA (eg by adding RNAse to remove RNA from the prep.). For ease, commercial kits are now available to shorten some of the steps and can be used for both small and large-scale isolation. ​ https://softchalkcloud.com/lesson/files/zxg6UA4wBGk1T8/Practical4_202021_print.html 3/13 11/23/23, 3:41 PM BIOL10401 Introduction to Laboratory Science Agarose gel electrophoresis Electrophoresis is a technique used to separate molecules according to differences in molecular size, and is based on the movement of charged molecules in an electric field. Agarose gels, derived from agar-agar (from seaweed), are used for the separation of nucleic acid molecules. The matrix of the gel acts as a molecular sieve, or a matrix of holes, through which smaller DNA fragments and RNA can move more easily than larger ones. The pore size of the gel depends on the concentration of agarose (typically 0.7 - 2 %), which can be varied depending on the size of the nucleic acid fragments to be separated. Higher percentage gels (> 1.5 %) allow separation of smaller fragments (< 1 kbp), whereas lower percentage gels (< 1 %) are more suitable for larger fragments (> 3 kbp). The agarose gel slab is placed between two electrodes in a chamber and immersed in a conductive buffer solution. Individual nucleic acid samples are applied to wells in the gel (Figure 4.1) and an electric current is then applied. At a pH of around 8.3, DNA is negatively charged and will thus move through the gel towards the positive electrode. Over a period of time, smaller DNA fragments will travel farther than larger ones. Fragments of the same size stay together and migrate as single bands. Electrophoretic separations can be performed vertically (gel standing upright) or horizontally (gel lying flat on a table). The agarose gel contains the dye SafeView, that binds to DNA and RNA and that fluoresces under long-wave UV light. The gel can then be viewed under UV light and photographed to see the nucleic acid. Agarose gel electrophoresis can be used to estimate the size of DNA fragments (see Semester 2 practicals for more details!) and for quantifying amounts of DNA in a sample. Figure 4.1 - A solution containing DNA is 'loaded' into 'wells' at the negative electrode end of the gel - an electric current is then applied and the DNA moves through the gel matrix towards the +ve electrode. Here is a video of how agarose gels are made and how they are loaded: (Unfortunately there are no subtitles available, but the images are useful) https://softchalkcloud.com/lesson/files/zxg6UA4wBGk1T8/Practical4_202021_print.html 4/13 11/23/23, 3:41 PM BIOL10401 Introduction to Laboratory Science Plasmid DNA on gels Interpreting gels containing plasmid DNA can be difficult. In addition to the plasmid DNA, contaminating chromosomal DNA and RNA may also be seen. To complicate matters further, plasmid DNA migration through an agarose gel depends not only on the size (molecular weight) of the plasmid, but also its molecular conformation. Plasmid DNA can exist in one of three major conformations: i) supercoiled The circular double-stranded plasmid DNA usually exists in the bacterial cell as a 'supercoiled' molecule (Figure 4.2), i.e. the DNA molecule coils about itself to form a compact structure (think what happens if you try to add twists to a loop of rope). This form is the fastest-moving in a gel. ii) relaxed or 'nicked' If one of the strands of DNA is 'nicked' or cut during the plasmid preparation, the torsional stress of the supercoiled molecule is released so that the plasmid develops into this relaxed open circle form (Figure 4.2.). This is the slowest-moving form of plasmid DNA in a gel, as the larger size impedes movement through the gel pores. iii) linear Linear plasmid DNA forms when both strands of the molecule are cut in the same place - either deliberately by a restriction endonuclease, or by accident during the extraction process. The linear form of the plasmid usually migrates at a rate in-between that of the supercoiled and the relaxed forms (but note that this is not always the case). Figure 4.2 - EM picture showing relaxed (far left) and supercoiled forms (remaining panels) Here is a diagram summarising where the plasmids run to on a gel: Quiz https://softchalkcloud.com/lesson/files/zxg6UA4wBGk1T8/Practical4_202021_print.html 5/13 11/23/23, 3:41 PM BIOL10401 Introduction to Laboratory Science https://softchalkcloud.com/lesson/files/zxg6UA4wBGk1T8/Practical4_202021_print.html 6/13 11/23/23, 3:41 PM BIOL10401 Introduction to Laboratory Science Experimental procedure for isolating plasmid DNA There is no laboratory session this semester to accompany this online practical, so go through the protocol as though you were performing it. You may want to write your own notes so you understand the steps involved. Two overnight broth cultures of E. coli are the source material for the plasmid preparations. Broth cultures A and B contain either the plasmid pBluescript or the plasmid pGLO. These plasmids are not the same size. Your task is to work out which culture has which plasmid in it. The following describes an alkaline extraction procedure. You could then use agarose gel electrophoresis to visualise the plasmid and to identify: 1. the different molecular conformations of plasmid isolated 2. the different species of nucleic acid that can be observed on an agarose gel 3. which culture has which plasmid 1. Overnight broth cultures A and B (1 ml of each) are added to a microcentrifuge tube (also called an 'Eppendorf tube', though this is a brand name!) The cultures are centrifuged for 1 min at high speed in the microcentrifuge (N.B. the microcentrifuges need to be balanced and needs the lid on the rotor otherwise it makes an awful noise) Microcentrifuge for spinning down volumes of less than 1.5 ml. Pelleted bacteria after the spin-down. This will pellet the bacterial cells so that you can remove the broth supernatant. Top tip: when placing tubes into the centrifuge, always orientate the tubes with the hinge of the cap of the tube sticking away from the centre. This means that any pellet will always form directly below the hinge in the tube (this is useful to know if your pellet is very small and hard to see!) 2.The broth supernatant can be poured off into a discard pot or disinfectant. To remove the last drops of liquid, the tube is inverted onto some tissue paper - this will absorb the liquid by capillary action - and discard the contaminated tissue in the autoclave bag. Care must be taken not to dislodge the pellet of cells, but they are usually stuck pretty firmly to the tube. 3. Ice-cold Solution 1 (200 µl) is added to the pellet, using the automatic pipette. The pellet needs to be dislodged from the bottom of the tube using the end of the pipette tip. By pipetting the liquid up and down, the cells can be resuspended in the solution. Solution 1 contains glucose, Tris and EDTA. Glucose is added to increase the osmotic pressure outside of the cells, so that they become vulnerable to rupture. Tris is a buffering agent, added to maintain a constant pH. EDTA binds ions https://softchalkcloud.com/lesson/files/zxg6UA4wBGk1T8/Practical4_202021_print.html 7/13 11/23/23, 3:41 PM BIOL10401 Introduction to Laboratory Science that are needed by enzymes that can degrade DNA. These enzymes ('DNases') are thus inhibited from degrading the DNA. 4. Solution 2 (400 µl) is added to the pellet-Solution 1 mix, and inverted 5 times to mix (gently - no shaking roughly). The suspension inside the tube should clear as the cells burst ('lyse'). Tubes are left for a minute on the bench to allow this to happen. Solution 2 contains sodium hydroxide and a detergent (SDS). The alkaline mixture ruptures the cells, and the detergent breaks up the lipid proteins of the cell membrane and solubilises cellular proteins. NaOH also denatures the DNA (both chromosomal and plasmid) into single strands. 5. Ice-cold Solution 3 (300 µl) is now added, and the tube inverted 5 times as above. The tubes are left on ice for 5 minutes. A white precipitate should now be visible: Solution 3 contains a mixture of acetic acid and potassium acetate. The acid neutralises the pH, allowing the DNA strands to renature. The salt precipitates the SDS from solution, along with all the cellular debris from the bacteria to form a precipitate composed of SDS, protein and lipid. The E. coli chromosomal DNA, which is now a partially renatured tangle, is also trapped in this precipitate (the large nature of the chromosome means that it does not rehybridise perfectly). The smaller, circular, plasmid DNA can escape the precipitate and remains in solution along with any RNA molecules. 6. The tubes are centrifuged for 5 minutes at high speed, to pellet the cellular debris: The cellular debris is pelleted to the bottom/side of the tube leaving the plasmid in the supernatant. 7. The supernatant (which contains plasmid) is transferred to a fresh microcentrifuge tube using a pipette. It is important here not to pick up any of the white precipitate during the transfer - it is better to sacrifice a little supernatant than to take any of this over. This precipitate contains the chromosomal DNA and we don't want any of that. The tube may need to be re-centrifuged if it is disturbed. 8. The centrifuge tube is now filled with iso-propanol (IPA; approx. 500 µl). The tube is inverted to mix contents, and incubated at room temperature for 2 minutes (no longer!) The IPA precipitates any nucleic acids (both RNA and DNA) from solution, and given time, will also precipitate proteins. https://softchalkcloud.com/lesson/files/zxg6UA4wBGk1T8/Practical4_202021_print.html 8/13 11/23/23, 3:41 PM BIOL10401 Introduction to Laboratory Science 9. The tube is centrifuged for 5 minutes at high speed. The nucleic acid may now be visible as a pellet at the bottom of the tube. However, be aware that a large pellet may indicate contamination with cellular components - a small or barely visible pellet may indicate a cleaner preparation, so big is not necessarily better! The supernatant is removed with a tip and discarded. 10. Ice-cold 70 % ethanol (1 ml) is added to the DNA pellet and washed by inverting the tube once, but without disturbing the pellet. A final centrifugation for 1 min at high speed will keep the pellet in the tube. Ethanol helps to remove any remaining salts and SDS. 11. Finally all of the supernatant is removed (without disturbing the pellet) using a tip. A tissue may be used to dry any visible ethanol if careful. The pellet is allowed to dry at room temperature. It is very important to remove all the ethanol (by flicking the tube to ensure there is none remaining) otherwise the DNA will not dissolve later. 12. When the pellet is dry (takes 10 min?), resuspend in 30 µl of TE buffer, and flick the tube to help dissolve the DNA pellet. TE buffer contains Tris and EDTA (see step 3). Pure DNA is very soluble in water, so if you ever have trouble dissolving the pellet, chances are it is mixed with other cellular components. We should now have extracted plasmid DNA! In order to visualise the DNA, the preparation should be run on an agarose gel. In this 'experiment' there are two strains of bacteria that have either pGLO or pBluescript plasmids in them. https://softchalkcloud.com/lesson/files/zxg6UA4wBGk1T8/Practical4_202021_print.html 9/13 11/23/23, 3:41 PM BIOL10401 Introduction to Laboratory Science Agarose gel electrophoresis - loading the gel A 0.8% agarose gel would be appropriate to see the plasmid DNA. The gel is placed into the electrophoresis tank and submerged so that the level of electrophoresis buffer is about 2 mm above the gel. The well-former (the "comb") needs to be removed if it is still in place. The comb being removed from the gel. The electrophoresis apparatus The gels will have SafeView or SafeStain added to visualise the DNA. It is a skin irritant so gloves are always worn when handling the gels. There are 8 wells in these gels. DNA markers ("M") are placed in Lane 1. These are molecular weight markers that can be used to calibrate the gel, and serve as standards to estimate the size of the unknown DNA fragments. A positive control is loaded in Lane 2 (this is previously purified pBluescript that has been cut in one place to produce a linear version of this plasmid). 1. The plasmid DNA sample is mixed with 5 µl of loading buffer. The loading buffer contains glycerol to ensure that the DNA samples sink into the wells in the agarose gel, and bromophenol blue, which acts as a marker dye to indicate the progress of electrophoresis. 2. Using a pipette, 15 µl of the prep./loading buffer mix can be loaded into one of the lanes of the gel. 3. The samples are electrophoresed at 80-100 V until the bromophenol blue has migrated to within 2 cm of the end of the gel. This takes about 45 min - 1 hour. The DNA can be visualised under UV light using a UV transilluminator and photographed. https://softchalkcloud.com/lesson/files/zxg6UA4wBGk1T8/Practical4_202021_print.html 10/13 11/23/23, 3:41 PM BIOL10401 Introduction to Laboratory Science Analysis of the Gel The photograph above shows a typical result of this experiment. The markers (lane M) are fragments of DNA with sizes 10, 8, 6, 5, 4, 3, 2.5, 2, 1.5, 1 & 0.5 kbp. Chromosomal DNA can sometimes contaminate the plasmid DNA preparation. Chromosomal DNA is much larger than plasmid DNA, and if present will be seen running higher in the gel than the 10 kbp marker fragment (it may run as a smear, if it has been sheared or degraded during the prep). There doesn't seem to any in this photograph. The linear plasmid (lane C) is approximately 3 kbp: supercoiled plasmid will run faster than linear, and for this plasmid, the relaxed circular form will run the slowest. Lanes A and B have the plasmid DNA from cultures A and B. Supercoiled plasmid DNA will always be present in plasmid mini- preps but the amount of the relaxed form varies between different preparations. RNA will always contaminate the plasmid DNA unless the enzyme ribonuclease (RNase) is used to degrade the RNA. Most of the RNA in the bacteria is smaller than plasmid DNA and single-stranded. It will run ahead of all the markers as a 'cloud' or bright smear. This bright smear may also contain smaller pieces of sheared DNA. Can you work out which plasmid (pBluescript or pGLO) was in which isolate? Quiz End of Online Practical 5​​ https://softchalkcloud.com/lesson/files/zxg6UA4wBGk1T8/Practical4_202021_print.html 11/13 11/23/23, 3:41 PM BIOL10401 Introduction to Laboratory Science Self-assessment of Online Practical 5 Check you have achieved the following specific biological learning outcomes: Learning Outcome Tick I understand how to prepare plasmid DNA from bacterial cells I understand the principles of agarose gel electrophoresis and how to interpret an agarose gel containing nucleic acid I can recognise the different conformations that plasmid DNA can adopt in agarose gels https://softchalkcloud.com/lesson/files/zxg6UA4wBGk1T8/Practical4_202021_print.html 12/13 11/23/23, 3:41 PM BIOL10401 Introduction to Laboratory Science End of Online Practical 5 Well done! You can go over the material in this online practical at any time. In order to register your completion of this resource, please take the quiz in Blackboard entitled 'Test your Understanding' Quiz of Online Practical 5 for which you need to obtain at least 60%. The quiz can be taken as often as needed to reach this total. This is accessed on Bb - Lefthand menu 'Test Your Understanding' or in the Content folder for Online Practical 5. The ILOs of this online practical are examinable in the BIOL10401 MCQ examination in January. If you struggle with any of the content, you can post a question on the Bb discussion site You can also discuss these activities in the PASS workshops (BIOL10401& DHS study group) Don't forget to complete any outstanding Data-Handling Skills Learning Modules and to attend the Data-Handling clinics if you have problems (see timetable) https://softchalkcloud.com/lesson/files/zxg6UA4wBGk1T8/Practical4_202021_print.html 13/13