RESTRICTION ENZYME DIGESTION LAB

Transcription

1 RESTRICTION ENZYME DIGESTION LAB Laboratory Overview 1) Set up restriction enzyme digestion reactions 2) Make predictions about results of restriction enzyme digestion reactions 3) Perform gel electrophoresis to analyze results of restriction enzyme digest reactions 4) Learn about the history of GFP usage in science Before Lab: Read Russell, Wolfe, Hertz, and Starr, Biology: The Dynamic Science. CH 18 Assignment (due at the beginning of next week s lab) Formal Lab Report on Restriction Digestion Analysis Overview: In today s lab you will use a technique called restriction enzyme mapping to determine the identity of two different plasmids (small circular pieces of DNA). One of these plasmids contains the coding sequence for the jellyfish gene product, green fluorescent protein (GFP), whereas the other plasmid does not. You will write a lab report detailing your findings from your restriction enzyme digestion reactions. Next week, you will take the plasmid that contains GFP-encoding DNA and introduce this plasmid DNA into E. coli bacteria via a process called transformation. In this manner, you will create transgenic E. coli that express the jellyfish gene product, green fluorescent protein (GFP), and hence glow green. Restriction Enzyme Digestion Lab, page 1

2 BACKGROUND RESTRICTION ENZYMES Restriction endonucleases (or restriction enzymes) are bacterial enzymes that act as defense mechanisms in these organisms. Restriction endonucleases cleave double-stranded DNA internally, cutting both strands at regions of specific nucleotide sequences that vary from one enzyme to another. The sequence cut by a restriction endonuclease is its target site (also called its recognition site). When foreign DNA, such as viral DNA, is introduced into a bacterial cell, a restriction endonuclease cuts the foreign DNA into shorter pieces, thereby interrupting most of the foreign genes. This helps defend the cell against invasion by and expression of genes that could be harmful to the organism. A bacterium protects its own DNA against digestion by its own restriction enzymes by chemically modifying its DNA soon after DNA replication, usually by adding methyl groups to bases within the target site of the endonuclease. The enzyme responsible for protection of the cell s DNA in this way is a DNA methylase. In molecular biology, restriction enzymes are used in several ways to modify and manipulate DNA molecules. One common use is to prepare fragments of DNA from one source to be combined with fragments of DNA from another source to construct recombinant DNA. Another use is to prepare small fragments suitable for nucleotide sequence analysis. Another use (which is what you will perform in today s lab) is to provide a rough map of the distribution of target sites for different restriction endonucleases. This is called constructing a restriction map. Constructing restriction maps are an important first step in analyzing a cloned DNA fragment or plasmid. By digesting the DNA with various restriction enzymes, alone and in combination, the number and relative positions of target sites along the DNA can be determined for each restriction enzyme. The resulting map can be used to determine information about the DNA being examined. In this exercise, you will use restriction enzymes to distinguish between two plasmids to identify the plasmid which contains the GFP coding sequence for use in next week s lab. Plasmids are small circular pieces of DNA that may contain coding sequences for proteins (eg genes). Some plasmids contain only one gene; others contain multiple genes. Often plasmids that have genes also contain regulatory sequences upstream of these genes, referred to as promoters. When these plasmids are introduced into bacteria, the bacteria will express the gene product downstream of the promoter because the promoter directs transcription of the DNA of the gene. After transcription, bacteria translate the mrna into the appropriate protein product. Next week, you will introduce the pglo plasmid into bacteria which will cause them to express the GFP gene product and glow green. Unfortunately, there was a mix-up during the preparation for this week s lab. Two different plasmids, pglo and pblu, were placed in separate tubes, but the tubes were not labeled. We now do not know which tube contains the pglo plasmid, and which tube contains the pblu plasmid. Luckily, the DNA sequence of both of these plasmids is known. In order to determine which tube contains which plasmid, you will digest each plasmid with the same restriction enzymes. Because the two plasmids have different DNA sequences, each plasmid will yield Restriction Enzyme Digestion Lab, page 2

3 different sized fragments after digestion. You can use the known DNA sequences of the plasmids to make predictions about the sizes of fragments that will be yielded after digestion with specific restriction enzymes. You can then compare your results to your predictions to determine the identity of each plasmid. In order to determine the sizes of your DNA fragments after restriction enzyme digestion, you will use a technique called gel electrophoresis. GEL ELECTROPHORESIS Gel electrophoresis is the most widely used method in molecular biology for separating macromolecules from one another on the basis of size. It is especially useful for analyzing mixtures of proteins or of nucleic acids with respect to the presence and relative abundance of molecules of different sizes, and for estimating the size of purified macromolecules; it can also be used as a step in purification of a specific protein or a specific length-class of DNA. Electrophoresis gels for nucleic acids are most commonly prepared with agarose or with polyacrylamide. In this laboratory, agarose gel electrophoresis will be used to separate DNA molecules that differ in number of nucleotides and, therefore, in length (size). With a few changes in details (gel preparation, buffers used, stains applied, etc.), the same overall approach would used be with a protein solution. The fundamental principle of gel electrophoresis is that charged macromolecules will migrate through a gel when an electrical field is applied across that gel. The distance migrated by a DNA molecule during the time it is subjected to the electrical field is determined by three factors: (1) the size (length) of the molecule, (2) the electrical field (dimensions and voltage differential), and (3) the density of the gel matrix. When a sample containing DNA molecules of different sizes is applied as a mixture to the same position in the gel, all of the molecules are subjected to the same voltage differential, and all of the molecules are challenged to work their way through the same gel matrix. This leaves molecular size as the only factor that will determine the final relative positions of the DNA molecules present in the original mixture. In environments of ph greater than 7, DNA molecules carry an abundance of negative charges that are uniformly distributed along the length of each molecule. In this environment, when DNA is subjected to an electrical field, the DNA molecules will migrate toward the positive pole of the electrical field. In a gel matrix, the movement of the DNA molecules in response to the electrical field is impeded by the gel, which acts like a sieve that slows the rate of their migration. The larger the molecule, the more difficult it is for the molecule to work its way through the gel; smaller molecules move through the gel faster than larger molecules. As smaller molecules get ahead of larger molecules, they also get closer to the positive pole, and their migration accelerates in proportion to their proximity to the positive pole: the closer the molecule gets to the positive pole, the faster it migrates. Consequently, as smaller molecules of DNA get ahead of the larger molecules, the relative rate of migration of the smaller molecules is determined by two factors: (1) their relative ease of working their way through the gel matrix, and (2) their increasing proximity to the positive pole of the electrical field. This compounding of influences on the rate of migration results in an exponential relationship between molecular size and distance migrated. Restriction Enzyme Digestion Lab, page 3

4 Gel electrophoresis results in the separation of a mixture of DNA molecules according to molecular length (size), with the smaller molecules moving ahead of the larger molecules. Wherever there are many DNA molecules of very nearly the same size, those molecules accumulate as a migrating band that is separated from smaller molecules ahead of them in the lane of their migration, and larger bands behind them. The positions of the bands can be visualized by staining the gel with a dye that binds to DNA molecules, and the size of the molecules in each band can be estimated by comparing the band s position (distance migrated) with the positions of DNA molecules of known size. In this technique, a standard curve serves as the means of estimating the size of the molecules. This is similar to the use of standard curves for estimating the concentration of smaller molecules, as you learned in Lab 2 (Spectrophotometry of Proteins) of this laboratory course. There is, however, one notable difference between the standard curves of Lab 2 and the standard curve that you will construct in this exercise. In Lab 2, the relationship between Absorbance and protein concentration (in the useful range of the standard curves) was linear. In this lab, the relationship between molecular size and distance migrated in an electrophoretic gel is exponential, as explained above. For this reason, the standard curve that you construct in this exercise will be plotted on semilogarithmic coordinates so that the curve generated will relate the logarithm of molecular size to distance migrated plotted on a linear scale. In order to perform gel electrophoresis, you will mix your RE-digested DNA samples with loading buffer. Loading buffer contains three important components: (1) a dye called SYBRgreen, which binds to DNA and fluoresces when exposed to short-wavelength light (~400 nm), (2) a dye called bromophenol blue which allows you to view the progression of your sample as it migrates down the gel, and (3) glycerol, which allows your sample to sink to the bottom of the well when you are loading the gel. To determine the size of the products from your RE digests, you will load a solution of DNA of known sizes (your markers ) onto your agarose gel along with your restriction enzyme digested samples. After the samples have been loaded into the gel, the gel will be covered with TBE buffer at ph 8. Then, the electrophoresis apparatus will be closed, electrical leads will be attached, and sufficient current will be applied to maintain a voltage differential of approximately 110 V across the gel. Electrophoresis will continue until the bromophenol blue indicator dye approaches the positive end of the gel (~ 1 hour). After the gel has been run, your instructor will disconnect the electrical leads and expose the gel to light of the appropriate wavelength to cause fluorescence of the SYBR green dye. A picture of this gel can then be taken, and the location of the fluorescent bands of the DNA markers can be measured. You can then use these measurements to estimate their length of your RE digestion products (in base pairs) by comparing the distance migrated by your sample to the distance migrated by the markers. Restriction Enzyme Digestion Lab, page 4

5 PROCEDURE 1A: RESTRICTION ENZYME DIGESTION 1. Sign out a micropipetter from your instructor. Practice pipetting 2.0 μl volumes. This is somewhat challenging to do accurately. Use the colored water on your lab benches to accurately measure 2 μl into one tube. BEFORE moving on to step 2, verify with your lab instructor or TA that the volume is indeed 2 μl. This is critical, because if you do not pipet accurate volumes, the RE digest will not work! 2. Each pair of students will perform one RE digest assigned by her lab instructor. Label one 0.5 ml tube, in which you will perform your assigned RE digest reaction. MA for MluI with template A; HA for HindIII with template A MB for MluI with template B; HB for HindIII with template B 3. Use the table below as a checklist while adding reagents to each reaction tube. Add components from left to right in the table. NOTE: The enzymes are in a glycerol solution that is viscous. It is important that you place only the very TIP of the pipet tip into the enzyme solution. If the tip is placed deep in the viscous solution, there will be more enzyme on the outside of the tip than within it. Also, too much glycerol in the digestion mixture can inhibit the activity of the restriction enzyme. water Buffer NEB 3 for MluI DNA template A or B Enzyme MluI or HindIII NEB 2 for HindIII 5 μl 2 μl 8 μl 5 μl 4. Pool and mix the reagents by tapping the tube bottom on the lab bench. Then spin the tubes quickly (~2 seconds) in the tabletop centrifuge in the lab. Your instructor will demonstrate use of this centrifuge. 5. Incubate all reaction tubes for 30 minutes at 37 o C. During this incubation time, make predictions about your expected results from the RE digest experiment (Procedure 1B). These predictions will be necessary for writing your lab report. 6. After 30 minutes, remove your reaction tubes from their temperature controlled area and proceed to Procedure C: Gel Electrophoresis. Restriction Enzyme Digestion Lab, page 5

6 PROCEDURE B: PREDICTIONS Generally, when performing RE digestion experiments, the first step is to make predictions about the expected sizes of DNA fragments after RE digestion. Due to time constraints, we will make our predictions after setting up our reactions; ideally, you would make your predictions before you performed an experiment. Below is a map of each plasmid: pblu and pglo. Because plasmids are circular, the number 1 is arbitrarily assigned to the first base of the origin of replication (labeled ori in the diagram below), and bases are sequentially numbered from that point. Genes (open reading frames) are diagrammed as blocks and promoter regions are diagrammed as arrows, indicating the direction of transcription. Even though all plasmids are composed of double-stranded DNA, only a single line is drawn to represent the double strand of DNA. Restriction enzyme target sites are diagrammed as lines and labeled with the name of the appropriate enzyme and the base-pair number at which the enzyme cleaves. The total size of the plasmid (in basepairs) is located in parentheses in the middle of the plasmid directly underneath the plasmid name. Note that pblu is 5,437 basepairs and pglo is 5,371 basepairs. pblu 1 MluI ori 632 pglo 1 ori amp R MluI 1057 amp R MluI 1135 HindIII 3190 pblu (5437 bp) ß-gal MluI 1837 pglo (5371 bp) gfp HindIII 1465 MluI 1666 HindIII 2114 In lab today, you will digest each plasmid separately with either MluI or HindIII. Note that these enzymes cleave at different locations along each plasmid, and thus will produce different sized fragments for each plasmid. Based on the sizes of the fragments produced after digestion with these restriction enzymes, you will be able to determine which tube (A or B) contains the plasmid pblu and which tube contains pglo. To determine the identity of the plasmids in tubes A and B, you will need to predict the expected sizes that will be produced after either pblu or pglo is digested with either MluI or HindIII. To do this, start with the pblu plasmid and the HindIII enzyme. Examine the diagram above. How many recognition sites are present for the HindIII enzyme on the pblu plasmid? Based on this information, how many fragments would you expect to be produced after digestion of the Restriction Enzyme Digestion Lab, page 6

7 pblu plasmid with HindIII? What are the sizes of these fragments? To help you with this task, you may wish to use the model kits found on your lab benches. Note that in these kits, each plasmid is represented by a set of tubing, and restriction enzyme recognition sites are represented by connector pieces. To simulate restriction enzyme digestion, remove the tubing pieces from each other at the connector sites. Use the diagrams and the model tubing kits to make your predictions. Record your predictions in the table below. In the first column of the table, write number of fragments produced by HindIII digestion, and in the second column, write down the size, in basepairs, of each fragment. Now examine the pglo plasmid diagram. How many recognition sites for the HindIII enzyme are present in pglo? If HindIII cleaved the pglo plasmid at all of its recognition sites, how many fragments would be produced? What are the sizes of these fragments? Use your model tubing kits and the diagrams to make your predictions, and record these in the table below. Note the different number and size of the fragments produced by HindIII digestion of pblu and pglo. Now, repeat this procedure for the MluI enzyme. Predict the number and sizes of the fragments produced by MluI digestion of the pblu plasmid. Predict the number and sizes of the fragments produced by MluI digestion of the pglo plasmid. Record these predictions in the table below. You will need these predictions to interpret your data for your lab report. Predictions for HindIII Digestion: # fragments Sizes of fragments (bp) pblu pglo Predictions for MluI Digestion: # fragments Sizes of fragments (bp) pblu pglo Before leaving lab today, check with your instructor to determine if your predictions are correct. This is a critical piece of information for you lab report. Restriction Enzyme Digestion Lab, page 7

8 Now that you know the sizes to expect from each digest, how will you visualize the DNA fragments after they are digested? To determine the size of each fragment, you will perform agarose gel electrophoresis on the products of the restriction enzyme digestion reactions. To do this, each restriction enzyme digestion reaction mixture will be mixed with loading buffer and placed into the wells of an agarose gel. An electric current will be applied, and the negativelycharged DNA fragments in each well will migrate toward the positive pole of the gel according to size. Smaller fragments will migrate faster and longer fragments will migrate slower. To determine the sizes of the fragments from your restriction enzyme digestions, you will need to compare the distance your unknown fragments migrated to the distance migrated by a known set of size standards, called a DNA ladder or DNA standard markers. Let s examine a sample agarose gel to help us understand how we will determine the sizes of the DNA fragments from our reactions. On the following page is a diagram of an agarose gel. There are eight lanes on this gel: lane 1 contains DNA markers (labeled standard), lane 3 contains sample B (labeled B) and lane 5 contains sample K (labeled K). The wells for each lane of the gel are noted by rectangles, and the DNA fragments are noted by lines. The smallest fragments have migrated the farthest and are thus located at the end (bottom) of the gel. A key for the set of standard markers is located on the left side of the gel. You can see that the markers contained a mixture of DNA fragments that were 20kb, 12kb, 7.5kb, 5kb, 3.5kb, and 1.5 kb. The 1.5 kb fragment, being the smallest, migrated the farthest. In order to determine the sizes of the fragments in lanes B and K, you must construct a standard curve using the distances migrated by the standards of known sizes. To do this, measure the distance traveled (in mm) for each of the standard fragments in the marker lane, and record your data in the table provided. Then, use the semi-log graph paper available in lab and plot the distance traveled (in mm) on the X-axis and the size of the DNA fragment (in kb) on the Y-axis (the log scale). Ask your instructor for assistance if you are not sure how to use semi-log paper. Draw a line of best fit connecting the points on your curve. Then, measure the distance traveled by each of the DNA fragments in lanes B and lanes K. Use your standard curve to determine what size each fragment is. Fill in the data tables on the next page with this information and have your lab instructor check that you have calculated this correctly. Understanding how to construct a standard curve using semi-log paper, and use it to calculate DNA fragment sizes is required for you to write your lab report. This is an example problem, meant to help you practice constructing standard curves. This example problem does not go in your lab report. Restriction Enzyme Digestion Lab, page 8

9 Example Predictions Assignment: Have your instructor check off that you completed this assignment before you leave lab today. Do not turn this in with your lab report. Fragment size (kb) 20 Distance traveled (mm) Fragment Lane B, #1 Lane B #2 Lane B #3 Distance traveled (mm) Fragment size (kb) Lane K #1 Lane K #2 Restriction Enzyme Digestion Lab, page 9

10 PROCEDURE C: GEL ELECTROPHORESIS 1. Using a micropipetter, add 5 μl loading buffer into your RE digestion tube. Mix the contents by gently lifting the mixture into the pipet tip and then expelling the mixture back into the tube only once. 2. Set your micropipetter to 15 μl. Place a fresh tip on the shaft, and load 15 μl from your RE digest + loading buffer mixture tube into your assigned lane on a 1.2% agarose gel. Each group member should write down which sample was loaded into which lane. 3. To determine the sizes of the DNA fragments generated from our RE digestions, we will compare their distance of migration with the distance of migration of DNA fragments of known sizes DNA markers. The instructor will load 6 μl of DNA markers onto one lane of each gel. 4. After all wells are loaded, the instructor will close the apparatus, attach the electrical leads to the power supply, and switch on the current. The power supply is regulated to deliver sufficient current to sustain a voltage differential of approximately 110 V across the gel, from the negative pole to the positive pole. Watch for tiny bubbles to rise from the electrodes, indicating that current is passing through your buffer/gel system. After about one hour, the indicator dye (bromophenol blue) should be approaching the positive pole end of the gel, and the current will be switched off and the electrical leads disconnected. 5. Your instructor will demonstrate for you the removal of a gel from the electrophoresis apparatus and placement on the visualization light box. Wearing latex gloves, she will lift the gel tray from the apparatus with the tray slanting downward at one end, where it will be supported by the instructor s gloved fingers. This tray, with the gel inside, will be placed on top of a light source to illuminate the SYBR green dye. Note: When removing gels from the electrophoresis apparatus, if the tray is not slanted toward the end that is blocked by the instructor s fingers, the gel is quite likely to slip out the other end of the tray onto the bench or the floor of the laboratory. This is invariably disappointing. PROCEDURE D: PREPARATION OF AGAROSE GEL 1. Because your time in the lab is short, the gel you and your partners use will be ready for you at the beginning of your lab period. Nevertheless, in order for you to learn the steps in gel preparation, you and your partners will prepare one gel that will be used by the next lab section. 2. Fit the gel tray into the casting tray. Make sure that the seal is tight. If you are unsure, ask your instructor a tight seal is critical! Insert the well-comb at one end of the tray. 3. Weigh 0.6 g of agarose using the balance, and place this into a 250-ml Erlenmeyer flask. Restriction Enzyme Digestion Lab, page 10

11 4. Measure 50 ml TBE buffer in a graduated cylinder. Pour the buffer into the flask and mix by gentle swirling. 5. Close the flask with plastic wrap and heat it for approximately 1 min. in the microwave oven. Watch the mixture as you microwave it to prevent it from bubbling over and spilling. Using a hot-glove to protect your hand, remove the flask and gently swirl the contents. Examine the contents to be certain that the agarose is completely melted; if not, return the flask to the oven for additional heating. When the agarose is completely melted, remove the flask to a water bath at 65 o C to cool for a few minutes. 6. When the molten agarose has cooled to 65 o C, swirl it gently to be certain that the agarose is evenly distributed, then pour the contents of the flask into the prepared tray. Rupture any air bubbles by poking them with a clean pipet tip while the agarose is still molten. 7. Allow the gel to stand until it appears firm and is slightly cloudy, then turn in the gel for use by the next lab section. PROCEDURE E: HISTORY OF GFP In 2008, the Nobel Prize in Chemistry was awarded for, the discovery and the development of the green fluorescent protein (GFP). Next week in lab, you will start a procedure to create transgenic bacteria that express the GFP gene product (e.g. the GFP protein), and hence glow green. The history of the discovery of GFP is rather fascinating, and learning about it will aid in your understanding of its importance in science. While your gel is running, work in groups of 3-4 and use the lab computers to learn about five important individuals who made significant contributions to the discovery and understanding of GFP. Answer the questions on the lab assignment. Hand in this completed worksheet before you leave lab today. PROCEDURE F: GEL ELECTROPHORESIS ANALYSIS 1. When electrophoresis is finished, inform your instructor who will help you bring your gel (in the tray) over to the gel documentation system, and set it up for picture-taking. Your instructor will take a picture of your gel as it is exposed to short-wavelength light. Each group member will receive a picture of the gel. Because of the fluorescence of the SYBR green dye, DNA bands will appear bright white and the gel will appear dark. 2. Take your picture back to your bench and find the bottom of the wells on the gel. Measure the distance from the bottom of the wells to each bright DNA band. Record these distances (in mm) in Data Tables provided on the following pages of this handout. Restriction Enzyme Digestion Lab, page 11

12 Use the following data tables to collect your raw data during lab. Use this information to help you in writing your lab report. Do not attach this version of the table to your lab report to use this table in your report, reconstruct it for your report. Remember that all tables and figures in your lab report need titles and captions. Data Table for Standard DNA (Markers) Agarose Gel Base pairs/molecule Distance from well to band, in mm 10,000 DO NOT INCLUDE 8,000 DO NOT INCLUDE 6,000 DO NOT INCLUDE 5,000 4,000 3,000 (brighter) 2,500 2,000 1,500 1,000 (brighter) (may not be visible) The solution of Markers that you loaded into agarose gel contained DNA molecules that varied in size from 250 to 10,000 bp (base pairs). There should be 13 bands in your Marker lane (although not all may be visible). Construct a standard curve for your gel; this will be an important figure in your lab report. Remember to write a title and caption for this figure. To construct a standard curve: a. Measure, in mm (estimate to 0.1 mm), the distance migrated by each of the bands in the markers lane. Measure from the bottom of the well at the beginning of the lane to the leading edge of each band. Record the distances in the Data Table for your agarose gel. b. For each band, plot the distance on the X-axis and the molecular size on the Y-axis. Semilogarithmic graph paper will be provided so that as the molecular size of each band is plotted, the points will appear on the paper in positions that represent their logarithms; it is not necessary to look up the logarithm of each molecular size. c. Draw a line of best fit through all the points that together clearly generate a straight line. Do not include the points for 10,000, 8,000, or 6,000bp on your graph. Restriction Enzyme Digestion Lab, page 12

13 Data Table for Restriction Mapping of Plasmids A and B Template Enzyme How many fragments? Distance (mm) each fragment migrated A HindIII Estimated size of Fragment size (bp) B HindIII A MluI B MluI Use your standard curve graph to estimate the size of molecules in any other band on the same gel: i. Measure the distance the band has migrated (in mm). ii. Find its distance on the X-axis, follow a vertical line upward until it intersects with the line of best fit, then follow a horizontal line toward the left to its intersection with the Y-axis. The value at which this line intersects the Y-axis is the size of the DNA molecules in the band. iii. Examine the lanes that were loaded with each of the digests, measure the distance (in mm) migrated by each of the discernible bands, and estimate the sizes of the molecules in each band (in bp) from your standard curve. Record these values in the data table above. If you wish to use this table for your lab report, do not attach this one instead, recreate this table (or a similar type of table) for your report. Don t forget to write a title and caption for this table in your report. iv. Keep your gel picture and attach it to your lab report, remembering to write a title and caption for this figure. Restriction Enzyme Digestion Lab, page 13

14 Restriction Enzyme Lab Assignment: The History of GFP Group Member s Names: Lab Day/Time/Instructor In 2008, the Nobel Prize in Chemistry was awarded for, the discovery and the development of the green fluorescent protein (GFP). Clearly, this protein has an important place in scientific history. Go to the website maintained by Marc Zimmer at Connecticut. College: Read the information on the home page. Then, click on the History link, located on the right side. Five individuals who were instrumental in the discovery and use of GFP in science are profiled. 1. Next to the name of these individuals below, list a description of their contributions to GFP (you do not have to write in full sentences). List between 1 and 3 accomplishments per scientist. 2. Place a star by the individuals who shared the 2008 Nobel Prize. 3. One of the Nobel Prize winners is a faculty member in the Department of Biological Sciences at Columbia! Circle this individual s name. 4. Turn in this worksheet before you leave lab today. Osamu Shimomura Douglas Prasher Marty Chalfie Sergey A. Lukyanov Roger Tsien Restriction Enzyme Digestion Lab, page 14

15 Restriction Enzyme Lab Assignment: Lab Report LAB REPORT GUIDELINES For this lab, you will write a formal lab report. Although you worked in a group to perform this lab, each individual should turn in her own assignment. This assignment is due at the beginning of lab next week. This lab report should be approximately 5-7 pages, double spaced (including figures, tables, etc). Remember a longer lab report is not necessarily a better one; good scientific writing is concise. Include the following in your lab report*: 1. Introduction: Background information to help your reader understand this study. Include the following: a. Brief introduction to restriction enzymes (REs) and their use in molecular biology. If you are writing more than a paragraph, you are probably writing too much. b. How will you use REs in this study (ie what is the purpose of this study and what are your hypotheses/predictions)? Note that you will not have a specific hypothesis instead, you will be performing an experiment to determine which of two possibilities is true. Since you did not have any data to guide you before you did this experiment, you should not make an arbitrary guess about which is true; instead you should make sure that your experiment will allow you to distinguish between the two possible outcomes. c. This is a lab report about RE digestion not GFP do not include information about the history of GFP and its usage in science in your lab report (even though you learned about it during this lab period). 2. Methods: Brief description of the methods you used: Be sure to include information about volumes and names of reagents used, incubation temperatures and times, etc. You do not need to include information about how you labeled your tubes, the size of your tubes, etc. This section will be short (~ 1 paragraph). 3. Results: For this report, your results section will be short. Include the following information. Don t forget to include information both in paragraph form and in tables (with legends/captions). a. The standard curve you prepared. Don t forget to include a title and figure legend/caption. b. The sizes (in bp) of your RE digest fragments (pooled from the class data of all four digests; calculated using your standard curve). You could put these values in a table. Remember, tables need titles and captions. Restriction Enzyme Digestion Lab, page 15

16 c. Your gel picture (with a title and caption). d. A paragraph or two explaining the results from the four different digests (make sure that in the text you refer to all figures/tables you present). 4. Discussion: Interpret your data. Include the following: a. Based on your data, which plasmid, A or B, is pglo and which is pblu? How confident are you in your conclusion and why? You may wish to refer to your standard curve in this discussion; however, there is likely no need to write more than one or two sentences about your standard curve in your discussion. b. Discuss the significance of this study (e.g. why is it important to know which plasmid is pglo and which is pblu? Hint: The answer to this question includes what you plan to do with the pglo plasmid in the coming weeks). c. Did you find anything surprising or unexpected (such as fragment sizes that were not predicted)? If so, why do you think you found these unusual results? 5. References. Cite all outside references if you used any. You should cite the lab manual and at least one additional outside reference (like a text book, reliable website, etc). Make sure that if you list a reference in this section, you have an in-text citation somewhere in your lab report. Never include direct quotations from your references; always paraphrase in your own words. Never cite Wikipedia it is not a reliable academic source. 6. Don t forget to include an informative title and your name/lab section with your report. *NOTE: The above instructions are meant as guidelines to help you prepare your report. They are, by no means, a comprehensive list of everything that should go into the report. Restriction Enzyme Digestion Lab, page 16