BIO 121 LAB 10 - DNA I

Transcription

1 BIO 121 LAB 10 - DNA I All cellular organisms store their hereditary information as the precise sequence of nucleotides in DNA, just as written information is stored as the precise sequence of letters on a page. Heredity therefore entails making an exact copy of each DNA molecule and ensuring that each daughter cell receives a copy. Much of the information stored in DNA specifies how, when and where to make particular proteins. Consequently, one can add hereditary information to a cell by inserting a new DNA molecule which specifies how to make a new protein, a process called genetic transformation. However, for this molecule to be passed on to the cell's offspring it must also contain instructions for its replication. Many bacterial cells contain small circular DNA molecules, called plasmids, which encode proteins that inactivate certain antibiotics. Plasmids also contain sequences, called origins of replication, which tell the cell how to replicate the plasmid and how many copies to make. Bacteria can therefore acquire resistance to certain antibiotics by taking up "drug-resistance plasmids." This week s lab is the first of two parts. This week we will extract drug resistance plasmids from bacterial cells, and we will make DNA in a test-tube using PCR (polymerase chain reaction). We will then analyze the DNA which we have made by gel electrophoresis. Next week we will test whether the plasmid DNA which we extracted is functional by using it to genetically transform other bacteria to become ampicillin-resistant and to make GFP (green fluorescent protein). We will also study the DNA which we extracted by restriction analysis. To keep the instructions from growing too large, this week I will only give you the instructions for today s activities, and next week you will receive the instructions for the remaining activities. However, don t lose your data sheet, because you will only get one data sheet for the whole lab.

2 Instructions for Bio 121 lab 10: DNA I page 2 DNA extraction and purification Although DNA is so vitally important, it is only a minor component of most cells. For example, only ~3% of the dry weight of E.coli is DNA. The remainder is mainly protein (~50%), RNA (~25%), lipid (~10%) and carbohydrate (~10%), along with various small molecules. Consequently, to study DNA it must first be purified. Purification entails separating DNA from the other cellular components; to do so we use certain unique chemical properties of DNA. We either selectively precipitate DNA while leaving other molecules in solution, or we selectively precipitate other molecules while leaving DNA in solution. After several selective precipitations we can reduce the contamination by other constituents from the initial 3200% (97/3) to perhaps 1 or 2%, a 1,000 fold purification. The first and often most critical step in purifying any macromolecule from cells is lysing (breaking open) them to release their contents. This is partly because many constituents are so fragile that it is difficult to lyse cells without damaging the contents, and partly because cells have enzymes which degrade macromolecules when released. Therefore, to extract DNA we must gently lyse cells (because DNA is easily broken by excessive force) in a buffer which inactivates the enzymes which degrade DNA. Fortunately, enzymes which degrade DNA are usually inactive at high ph and also require magnesium as a cofactor. Therefore, the first step in most procedures for isolating DNA is to remove the cell wall (if present), then dissolve the cellular membranes with detergent in a buffer which inactivates the deoxyribonucleases. This is usually accomplished by keeping the ph high and by adding a chemical which chelates (binds) any magnesium ions present. Next, many contaminating proteins are denatured by raising the salt concentration, and the resulting precipitate is removed by centrifugation. DNA is then recovered from the supernatant by making use of the fact that it can be selectively precipitated from salty aqueous solutions by organic solvents such as ethanol. If necessary, further purification can be achieved by other selective precipitations, by treatment with enzymes which destroy contaminating RNA and protein, or by treatments which use other characteristics of DNA such as its density to separate it from other cellular constituents. PCR (Polymerase Chain Reaction) PCR is a technique for making many identical copies of a specific DNA sequence in a test-tube. It relies on the fact that all DNA polymerases work by adding one base at a time to a primer, using the complementary strand to determine which base to add next. Therefore, one can amplify a specific sequence by denaturing a fragment of DNA containing this sequence in the presence of DNA polymerase, deoxyribonucleotides and two primers, one which anneals to the bottom strand at the start of the sequence, and another which anneals to the top strand at the end of the sequence. Upon renaturing, the first primer will anneal to the bottom strand, and the second will anneal to the top strand. DNA polymerase will then add to each of these primers, using the complementary strand to decide which base to add next. It will thus create a new copy of each strand of DNA, beginning at the primer. The actual sequence of events goes like this: 1. DNA containing the sequence to be amplified is briefly heated to separate the strands. 2. It is then cooled to allow primers to anneal to each strand 3. DNA polymerase then extends the primers, using the complementary strand to determine which base to add next. 4. The reaction mix is briefly heated to separate the strands (and end DNA synthesis). 5. The entire cycle of heating, annealing, and synthesis is repeated 25 to 40 times. Upon completing 25 to 40 cycles you will create a fragment of DNA which starts at one primer and ends at the other. More information about PCR is given on pp. 269 of your text, and at or DNA analysis by gel electrophoresis

3 Instructions for Bio 121 lab 10: DNA I page 3 Size of a DNA molecule can be determined by a procedure called gel electrophoresis, where molecules are drawn through a gel-like matrix by an electrical field. In this procedure a gel forming a molecular mesh is suspended such that its ends are in contact with separate buffered salt solutions. One end of the gel is connected to the positive (red) electrode and the other is connected to the negative (black) electrode of an electrical power source, then a current is applied which flows through the gel. Molecules added to the gel are drawn towards whichever electrode is opposite to their own charge. Since DNA has a negative charge, it migrates towards the positive electrode (run towards red!). The rate of migration is inversely proportional to its size and shape, because bulkier molecules thread their way through the mesh more slowly than smaller molecules (just as a human can't run through a hedge as rapidly as a rabbit). Molecular weights of particular molecules can then be estimated by comparing the distance they migrated with the distance traveled by molecules of known molecular weight. DNA is usually measured in number of base-pairs rather than molecular weight; to convert length to molecular weight multiply by 660 Daltons. Samples are usually loaded into rectangular wells in the gel, so after electrophoresis we usually find that each sample is resolved into one or more rectangular bands which have traveled varying distances from the origin. More information about gel electrophoresis is given on page 269 of your text, and at Experimental Procedure Molecular procedures often require waits between steps. For example, the PCR reactions must cycle 25 times before you can analyze the products. Similarly, the lysozyme must digest the cell walls before you can lyse them. Therefore, you will need to start one procedure, then start a second while the first runs its course, and it will be important to track your progress through the various procedures. 1) PCR You will use PCR to amplify the DNA sequence encoding GFP from plasmid pglo, in order to ensure that the plasmid which you started with was correct, and a different DNA sequence from a control plasmid (which I know works well from previous experiments). You will be provided with plasmids containing the sequences to be amplified, primers which anneal to either end of these sequences, and with DNA polymerase and deoxyribonucleotides. You will then set up the reactions, run them, and determine the size of the products by gel electrophoresis. 1. Prepare 2 PCR reactions as follows. Change your pipet tips after every transfer! a. obtain 2 of the tiny 200 µl PCR tubes b. draw a red ring around one and a blue ring around the side of the other, then place both on ice. c. add 1 µl PCR buffer to each. d. add 2 µl T3 primer to each. e. add 2 µl T7 primer to each. f. add 4 µl 1 mm dntp mix to each tube. g. add 1 µl pglo to the red tube, and 1 µl control plasmid to the blue tube. h. give both tubes to your instructor, who will add 10 µl taq DNA polymerase stock to each tube. 2. Once everyone is ready, your instructor will run the PCR reaction: First the samples will be kept 2' at 94 to denature the DNA, then you will run 25 cycles of 94 (to denature the DNA), 55 (to allow the primers to anneal to the templates), and 72 (to allow taq DNA polymerase to synthesize the new strands). 2) DNA extraction You will be provided with cultures of E. coli which were transformed with pglo and grown in liquid medium supplemented with ampicillin. These cells were then harvested by centrifugation and resuspended in a lysis solution buffered to ph 8.0 by Tris hydrochloride (Tris Cl) and that contains a chemical called EDTA which chelates Mg 2+. To extract their DNA you must first lyse these cells by digesting their cell walls with an enzyme called lysozyme, then dissolving their plasma membranes under alkaline conditions with a detergent called sodium dodecyl sulfate (SDS). You will then precipitate proteins and other debris by raising the salt concentration to over 2.5 moles/liter. Plasmid

4 Instructions for Bio 121 lab 10: DNA I page 4 DNA will remain dissolved in the liquid phase, so the precipitate can be recovered by centrifugation. You will then precipitate the DNA by transferring the supernatant to a fresh tube, adding two volumes of ethanol and harvesting the precipitate which forms by centrifugation. Next week you will examine the DNA which you extracted by digesting it with restriction enzymes, and by using it to genetically transform bacteria to make GFP and inactivate ampicillin. 1. Do not begin until you have started your PCR reactions. 2. Obtain a tube containing 90 µl of E.coli transformed with pglo and resuspended in lysis buffer (50 mm Glucose, 25 mm Tris Cl ph 8.0, 10 mm EDTA). 3. Add 10 µl lysozyme solution (2 mg/ml in lysis buffer) to the tube, mix by gently swirling, then leave 10 minutes at room temperature to digest the walls. Your cell suspension should look opaque. 4. Add 200 µl of 1% SDS in 200 mm NaOH to the tube, and mix by gently rocking the tube. Be gentle, you are lysing the cells! You should see the solution clear and become more viscous as the cells lyse. Continue rocking gently until the solution clears completely. 5. Add 150 µl of 10 M ammonium acetate and place on ice. You should see a white precipitate consisting mostly of protein form almost immediately. 6. Recover the precipitate by centrifuging for 10 minutes. Be sure to balance the rotor by placing another tube directly across from yours in the centrifuge. 7. Transfer the supernatant to a microcentrifuge tube labeled with your name containing 1000 µl of ethanol. A white precipitate should form almost immediately. This is your DNA! 8. Leave your tube on ice for 10 minutes, then recover your DNA by centrifuging for 10 minutes. 9. Remove as much ethanol as possible without disturbing the pellet, then place the tube on its side and leave it to dry for 5 minutes. 10. Add 70 µl TE to the tube, then flick it a few times to mix the DNA and buffer. Be sure that your tube is labeled so that you can recognize it, then place it in a rack to be stored until next week. 3) Gel electrophoresis You will determine the size of your PCR products by electrophoresis through a gel made of agarose (a complex mixture of carbohydrates extracted from seaweed), and comparing the distances migrated with the distance migrated by standards of known size. 1. After adding lysozyme to your cells prepare 1.5% agarose gels for electrophoretic analysis of your samples. a. Set up a gel bed with a 6-well comb as demonstrated by the instructor. b. Obtain a tube containing melted 1.5 % agarose in electrophoresis running buffer. Pour the gel into the prepared gel bed and allow the gel to harden (~30 min.). c. Remove the combs from the gel, being careful not to tear the wells. Release the "gates" from the gel bed and place it into the electrophoresis chamber. Add electrophoresis buffer to the chamber until it covers the gel completely. 2. Once the PCR run is complete and you have obtained your samples, put on a pair of gloves and keep them on for the rest of the lab! Add 4 microliters of loading dye to each sample. The loading dye contains the fluorescent dye EZ-Vision to enable us to see and photograph the DNA. Since it binds DNA, it may be carcinogenic! 3. You will run one gel/pod, therefore one group will load 10 µl of their red sample into lane 1 (the lane closest to the low side of the gel box) and 10 µl of their blue sample into lane 2, while the other

5 Instructions for Bio 121 lab 10: DNA I page 5 group will load 10 µl of their red sample into lane 4 and 10 µl of their blue sample into lane 5. Your instructor will load 10 µl of a molecular weight marker sample into lane 3. Save your leftovers in case the first batch messes up. 4. Attach the electrophoresis chamber to the power supply and electrophorese until the blue dye band is about 2/3 of the way down the gel. For our system, this is typically 100 volts for 60 min. 5. After electrophoresis is completed, turn off the power supply and disconnect the chamber. 6. Photograph the gel as instructed. 7. Prepare a standard curve of log bp vs. distance traveled for your size standards, and use it to estimate the size of the PCR fragments which you obtained. Your size markers are obtained by digesting a cloned gene from my collection with Dde I. The fragments are 1107, 695, 540, 409, 310, 240, and 166 bp long. 8. Write up the PCR experiment in the usual format. Your introduction should explain what PCR is, why it can be used to amplify a specific fragment of DNA, and the purpose of this experiment. Your results should include a table of distance traveled and size of the standards, and distance traveled and estimated size of the PCR products, a graph of log bp vs. distance traveled, and sentences explaining the purpose of the experiment, where the data are presented, and what the key results were. Your discussion should explain why you obtained the bands observed in each lane, and why the two bands differed in size.