Writing Lab Reports General Comments The objective for scientific report writing for this course is to teach you how to prepare Figures and Tables that are notionally suitable for submission

to a scientific journal for publication. The style that will be used is partially based upon the American Chemical Society (ACS) journal Biochemistry and is described in detail below. You are NOT required to write complete journal type scientific reports for the experiments that you will perform this term. Instead, you will analyze your data and prepare any graphs or tables that are required. You should be aware that preparing titles and captions for Figures and Tables is somewhat subjective and that different levels of detail can be found between different articles in the Biochemistry journal. However, for purposes of standardization and marking fairness you are required to adhere to the format provided in the relevant sections below even if you can find contrary examples in Biochemistry. No sample calculations are required for your reports Collecting and Reporting Data Recording Data Students are expected to record their data as they collect it. There are two places for data to be recorded: 1. Within the body of the protocol, and 2. In the data record sheet that can be found at the end of each experimental protocol. The reason for organizing data recording in this manner is so that you can record your data in your manual while working through the experimental protocols without having to flip pages to do so. The recording of data in the data record sheet is done as you must submit a picture of this data sheet with your submitted lab reports so that the marker can verify that information entered into the excel data sheet that you submit to your submission folder is correct. You are required to complete your data record sheet prior to leaving the lab as you are required to have it signed by your TA before departing. Significant figures Any measured value has error, and significant figures are used to provide information about the precision and uncertainty associated with any measurement and calculations based upon them. The number of significant figures in a number can be unambiguously indicated through the use of scientific notation, and the number of significant figures that are to be used when reporting a value is determined by the equipment used to make the measurement. (See the discussion above regarding recording data-remember the last digit on the right indicates the level of certainty about a measurement). Determining the Number of Significant Figures (All numbers in a number are significant unless they are zeros. Zeroes may or may not be significant according to the following rules: 1. Zeros are always significant when they are found in the middle of a number. e.g. 407.02 has five significant figures. 157 2. Zeros are significant when they are on the end of a number on the right hand side of a decimal place. e.g. 39.50 has four significant figures; 44.00 also has four significant figures. 3. Zeros between a decimal and a number on the right hand side of a decimal are not significant. e.g. 0.004 has only one significant figure; 0.0040 has two significant figures 4. Zeros at the end of a number but to the left of the decimal are not significant but they generate ambiguity. e.g. 50 has only one significant figure. If you wanted to indicate the number 50 to two significant figures you would have to use scientific notation and write it as 5.0x 10. However, it will be unclear to the reader whether you meant to indicate one or two significant figures, so to eliminate uncertainty always use scientific notation in these situations. Calculations with Significant Figures When you are performing calculations with your data, execute the calculation using all available digits from the equipment you are using (even if you are using numbers with different numbers of significant figures). However when you report the final answer for the calculation you must use the number of significant figures that are appropriate for the type of calculation or marks will be deducted. The rules for performing addition and subtraction calculations with significant figures are different than the rules used for multiplication and division. You must report your data according to the correct set of rules. Addition and Subtraction The number of significant figures is determined by the number of decimals places in the least precise number. This may result in your answer having more or fewer significant figures than the original numbers that were used for the calculation. e.g. 14.421 has five significant figures and is precise to the third decimal point. 0.21 has two decimal points and is accurate only to the second decimal point. If 14.421 is added to 0.21 your answer before taking into account significant figures is 14.631. To report this value using significant figures you report 14.63 to indicate that the uncertainty lies in the second decimal point. If you subtracted 3.22 from 3.68 your answer would be 0.46. The answer only has two significant figures even though your original numbers each had three significant figures. Multiplication and Division The number of significant figures is determined by the number of significant figures in the number with the fewest significant figures. e.g. 6.332 multiplied by 2.40 equals 15.1968, but to report this value using significant figures you would report 15.2 since there are only three significant figures in the number with the least number of significant figures. 158 Presenting Experimental Data as Figures Note: This section will focus upon preparing graphs since most of the figures that you will prepare this term will be graphs. Background Presenting data in a graphical form is intended to give a clear presentation of the results that were obtained for a particular experiment or experiments. Graphs should be self- explanatory and this means that a person with relevant scientific knowledge should be able to look at the graph and understand what it means without reading the entire report. You must use computer graphing software to prepare graphs. You must show your data points on the graph and one of the following types of lines must be present: (i) a straight line, (ii) a smooth curve, or (iii) each point joined together. The particular situation will dictate which type of line is to be used. A best-fit line and its equation of the line is required for calibration curves or when a linearization of graphed data is required. Place the equation of the line on the graph next to the best-fit line. When you use a best-fit line DO NOT also join the points with a second line. A calibration curve is not forced through zero. Column graphs will be used only when you are directed to do so. Axes The ordinate (y-axis) should be used for the dependent variable and the abscissa (x-axis) for the independent variable. (NOTE: dependent is measured, independent is chosen.) The axes should be clearly labeled and the correct units provided in brackets immediately thereafter. The axis label should unambiguously identify its variable. In the example graph provided by Figure 45 on the next page, data was obtained by measuring the absorbance of a solution of 4-nitroaniline at different wavelengths. Absorbance was measured so this variable belongs on the y-axis, and the wavelengths were chosen, so they by default belong on the x-axis. Absorbance and wavelength do not require further explanation as their meaning is well-understood by a scientific reader. Absorbance has no units and the units for wavelength are nm. Figure Caption Each graph must be accompanied by a figure caption that is found below the graph. A figure caption has several components, (see sample figure caption provided below): 1. Figure number 2. Figure title 3. Additional Information The totality of the information in the figure caption should answer the following questions: a) What type of data is being depicted? If the figure is a graph you should indicate what type of graph it is, e.g. calibration curve, binding plot etc. b) What compounds/organisms etc. were used to gather the data from which the graph was prepared? c) Under what conditions or in what? For example, if the experiment was performed in a buffer, identify the buffer in full. If a special reagent was used, identify it. 160 Understanding and Using Dilutions Basic calculations Understanding dilutions is an important concept for anyone working in a laboratory. Typically, dilutions are required to ensure that measurements are within the reliable recording range of the instrument/technique that is being used to investigate a problem, or to lower the concentration of stock solutions to a working concentration. Dilutions will be expressed in this lab manual as Fold Dilutions or as a dilution factor which refers to how much the solution of interest has been diluted. Fold dilution can be calculated as follows: fold dilution = final dilution volume original volume of what you are diluting Example 1: if you diluted 15 mL of 0.1 M substance X by adding 85 mL of water the fold dilution would be 100 mL/15 mL which equals a 6.67 fold dilution. If you want to calculate the new concentration of substance X simply divide the original concentration by the fold dilution and you will have your answer. This last sentence can be expressed as: original concentration fold dilution 3.53 new concentrat ion - So in this example the final concentration of substance X after you diluted it is 0.015 M. Example 2: if you were instructed to make 100 mL of a 20 fold dilution of solution Y, just set up the first equation above to solve for the original volume of solution Y that you need. The original volume you need would be 100 mL/20 fold dilution which equals 5 mL. So you would mix 5 mL of Y with 95 mL of water/buffer. Example 3: Let us say you made a calibration curve for some analysis that ranged from 0.1 mg/mL to 1 mg/mL, but when you analyzed a sample from your experiment you obtained a reading that was much higher than the range of your standards. Since you do not know how your calibration curve behaves outside the range you have you do not know for sure the concentration of your sample. So if you took 1 mL of your sample and diluted it 5 fold, (you added 4 mL of water or buffer to your sample and mixed it), and then obtained a new reading of 0.42 mg/mL you would not be done because this is not the concentration of your sample but the diluted concentration. To obtain the original concentration you would multiply the new concentration (0.42 mg/mL) by the fold dilution (5 times) and you would now know the original concentration of your sample is 2.1 mg/mL. Multiple dilutions If you are performing a series of dilutions, the dilution factor for each step is calculated according to the examples provided above. However, the overall dilution for a sequence of dilutions of one analyte is the product of each of the individual dilution factors. Example: If a solution of substance X was diluted by mixing 5 mL of it with 95 of ethanol, and then 10 mL of the substance X and ethanol mixture was diluted with 90 mL of water, the total dilution of the substance X in the water would be 200 fold. This is because the first dilution was 20 fold (100/5) and the second dilution was 10 fold (100/10), and the product of 20 x 10 = 200. If you knew the original concentration of substance X was 10 mM, the final concentration would be 0.05 mM or 50 μM. 159 the number (i.e. don't use bold or italics). Number the figures using Arabic numerals (1, 2, 3 etc.). Follow the number with a period (i.e. don't use a colon). 2. Figure title The figure title should be a brief informative description of what the graph depicts or is about. It is preferable that the figure title is in non-sentence format. The title is where the type of graph (if there is a technical name for it) and the principal subject matter of the graph should be identified-it should not be just a repeat of the labels on the axes. For example, the graph above is obtained by measuring the absorbance of a solution of 4-nitroaniline at different wavelengths. Absorbance versus wavelength is not an appropriate title. An example of a more appropriate title for such a graph might be "Absorption spectrum of 4-nitroaniline." 4- nitroaniline is the subject of the graph and the phrase "absorption spectrum" implies to the scientifically-trained reader a particular relationship in this case the effect of changing the wavelength on the light-absorbing properties of the solution. Information about the various conditions under which this data is obtained is of secondary importance and is not title worthy and therefore is relegated to the additional information portion of the figure caption. For some graphs there may be multiple subjects all of which are title worthy. If there are only two subjects, identify them explicitly in the title, but if there are more than two, use a more generic group name for them and then identify them explicitly in the additional information or legend. For example, you might have a graph that looks at some property of various amino acids; if there were only two amino acids used, name them in the title, but if there were three or more it would be better to just use the word amino acid in the title rather than listing them all out. When you are using generic titles, still try to be specific as possible. For this amino acid example, it would be better to use "amino acids" as the generic group name rather than say "organic molecules". Using "organic molecules" while not technically incorrect as a group name, is too vague and would not really give the reader much insight into what the graph is about. Remember, graphs (and tables too) are intended to convey information in a concise and succinct manner. 3. Additional information You will frequently find that axis labels and the figure title do not provide sufficient information about the graph for an educated reader to understand it, or be aware of important information about it and thus additional information is usually added. The additional information portion of the figure caption is separated from the figure title by a period. The additional information may be a single or multiple sentences depending upon the requirements of the figure. Information that belongs in this part of the figure caption is all available information, which if different, might or would change the results presented in the graph. To elaborate on this point based upon Figure 45, you know that concentration affects absorbance so indicating the concentration of the 4-nitroaniline is crucial. What other compounds are present will often affect measurements, so the final working concentrations of any other substances that are present must be given. In our working example, there is only a buffer, so we give its full final concentration and pH. You will also know that cuvette path length affects absorbance, so specifying the path length is important, but because all the cuvettes used for this course have a path length of 1 cm and we are limiting you to 5 lines for your figure caption, path length will not be a required component of your additional information. 162/n Experiment 5: Purification of Spinach Genomic DNA and Agarose Gel Electrophoresis Analysis of Lambda DNA Restriction Digests Introduction to molecular biology Since the 1960s the development of the collection of methodologies commonly known as molecular biology has revolutionized the fields of biochemistry, cell biology, evolution and genetics. In particular, techniques such as the polymerase chain reaction (PCR) and DNA cloning have made it possible to study in great detail the molecular basis of the relationship between structure and function in many biological molecules. Molecular biology techniques have also been used to begin to unravel the intricate signalling networks that enable individual cells, tissues and organisms to cope with the dynamic challenges of their environment. During this week you will learn about and perform several of the core methods used in molecular biology extraction of DNA from a tissue; digestion of DNA using restriction enzymes; quantification of purified DNA by absorbance spectroscopy; and analysis of purified DNA by agarose gel electrophoresis. DNA Purification, Restriction Enzymes and Molecular Cloning DNA Purification control. P The first thing that a researcher needs to do if they want to analyze or manipulate DNA is to find a way to get it out of the cells that contain it, and then to purify it to ensure that there are no contaminating molecular species present that may distort experimental results or interfere + with any subsequent reactions (PCR, digestion, ligation etc.) to which the DNA might be subjected. Historically, DNA extraction and purification procedures utilized potentially hazardous organic solvents with complicated, delicate procedures that were difficult for less experienced researchers to replicate. Presently, well-equipped modern molecular biology labs make extensive use of commercially prepared kits that have simplified and standardized the extraction and purification of DNA that make it relatively easy for novice experimenters to obtain good yields of pure, high-quality DNA. In the simplest terms, after the cells of the tissue have been lysed, the kits work by selectively degrading and precipitating non-DNA-biological molecules which are removed by centrifugation or by washing them off a column. The columns for DNA purification are usually silica-based, and DNA molecules will bind tightly to these columns under certain ionic conditions--this allows for the removal of residual contaminants without significant loss of DNA. The DNA can then be eluted out of the column by altering the salt and pH conditions in the column. At this point the DNA can be used immediately for further investigations, or stored at -20°C until required. (column Evaluating the effectiveness of DNA purification When one is isolating or manipulating DNA, (in your case extraction and purification), one needs to determine if the procedure has worked properly, and this means of course that the DNA sample will require some form of analysis. The two most important factors for evaluating DNA quality are purity and structural integrity. The quickest and easiest way to evaluate the DNA purity is by measuring the absorbance of a small volume of the purified sample at 260 and 280 nm. DNA absorbs light well at 260 nm, whereas protein absorbs well at 280 rim (you saw this in your previous experiment), so if the 260/280 absorbance ratio is high, it suggests the DNA is relatively puré. Normally a value for this ratio between 1.8 at 1.9 is desired. However, even if one has successfully obtained pure DNA, it does not mean that it is high quality 85 Loryne 9201 A DNA sample could be pure (ie. free of protein), however, it may not be nigh quality i it is extensively degraded. Fragmentation of eukaryotic genomic DNA during purification is inevitable as the removal of histones and other stabilizing proteins leaves the DNA very fragile, and it invariably degrades into smaller pieces. This itself is not problematic so long as the average pieces are relatively large-say larger than 10 kilobases, because pieces of this size will leave numerous copies of individual genes and regulatory DNA intact, and therefore the DNA sample can be still be used for whatever purposes it was originally purified. If the DNA were smashed into much smaller fragments, it would be much less likely to contain intact pieces of desired sequences, and therefore unlikely to generate good results in the subsequent stages of a longer experimental protocol. Since size is one of the properties of DNA that can tell a researcher something about its quality, having a method that can be used to quickly and cheaply determine the approximate size of DNA is a useful tool to aid the evaluation of DNA quality. One of the simplest and most widespread methods to analyze DNA size is to perform agarose gel electrophoresis upon a few microliters of the sample. Restriction enzymes and molecular cloning One of the most significant technical advances in the history of biochemistry was the development of methods that allow researchers to transfer a small portion of DNA (say a gene of interest) from one DNA molecule into a different DNA molecule and then making numerous /\ copies of the new hybrid molecule. This process of recombining DNA is known as molecular cloning. We will not consider molecular cloning further in this course as it is outside the scope of our objectives, but the ability to do cloning requires the ability to cut DNA in a very precise manner. This ability to cut out a piece of DNA is dependent upon a specialized group of enzymes found in bacteria known as restriction enzymes. Restriction enzymes are proteins that cut the sugar phosphate backbone of DNA only when a specific sequence of nucleotides is present. For example, the restriction enzyme Xhol only cuts between nucleotides C and T of the sequence iausixe oplem 5' CITCGAG 3' 3' GAGCTIC 5' og nitido When a piece of DNA is incubated in the presence of a restriction enzyme, the enzyme will cut the DNA wherever it finds its target sequence, and therefore a series of fragments will be produced depending upon the specific sequence of the DNA and the restriction enzyme used. This incubation and cutting process is known as "digestion", and it is the precise control over the restriction digest fragments that enables experimenters to assemble different pieces of DNA from different DNA molecules into a new DNA molecule with the desired nucleotide sequence. In the lab for this week, you will observe how different fragments are produced when different restriction enzymes are used to cut the same sample of DNA. Electrophoresis Basic Principles DO +1- When charged molecules are placed in solution and exposed to an electrical field, the molecules will have the tendency to migrate to the pole opposite to their charge. This is illustrated in the picture below depicting how negatively charged molecules migrate towards the positively charged anode, whereas the positively charged molecules will migrate towards the negatively charged cathode. Anode doo bo Figure 23. Direction of charged particle movement during electrophoresis. Cathode V= This process can be exploited to separate charged molecules, and not surprisingly, has been extensively utilized in biochemistry and related fields for a wide variety of useful applications which are grouped together under the rather generic name of electrophoretic techniques. 1 Electrophoresis can be described as the examination of the movement of charged particles through solution under the influence of an electric field, and apart from the fact that oppositely charged molecules move in opposite directions, one of the most useful attributes of electrophoresis is the fact that the speed at which a molecule moves towards the oppositely charged pole, the migration velocity, is different for different molecules. The migration velocity of any particular molecule is dependent upon a variety of factors which can be very simply represented by the equation: speed Eq f (1) where v is the velocity of the charged particle; E is the strength of the electric field to which the charged particle is exposed; q is the net charge on the particle, and fis the frictional coefficient of the particle as it moves through the solution. 加速 When you apply an electric field (E) to charged molecules in solution they will start to accelerate towards the pole of the opposite charge. How fast they accelerate depends upon both the strength of the field (E), and how much charge (g) the molecule carries. However, as the molecule begins to move faster through solution the viscous drag that results from moving through solvent molecules also increases, and this drag is influenced by the size and shape of the molecule. The viscous drag is a force of opposite direction to that of the migrating particle. The size and shape of the molecule is represented by the frictional coefficient (f). Thus, the overall of = V (Steady stute velocity effect is that almost immediately after the voltage is applied and maintained at a constant level, the force accelerating the molecule forward, (Eq), is equalized by a force. (. decelerating it, and a STEADY STATE velocity or migration speed through the solvent is attained. This steady state velocity is denoted by the term from the above equation. It should be obvious upon examination of equation 1 that in any specific electrical field that the E is the same for all molecules in that field, and therefore the migration velocity of any particular molecule will depend upon its q/f ratio. This is sometimes mistakenly referred to its "charge-to-mass ratio", but in the context of solution-based electrophoresis, the magnitude of fis extremely complex and as indicated in the previous paragraph, is determined in part by the size and shape of the charged particle. We will not delve into the details of how the frictional coefficient can be modelled, and instead our attention will turn to the application of the basic principles of electrophoretic theory to practical considerations for the electrophoresis of nucleic acids. Electrophoresis of DNA The picture below is a representation of the structure of DNA at a neutral pH, and the structural element that is most relevant for electrophoresis is the regularity of the negatively charged phosphate groups (yellow circles) along the length of the structure. H₂C 03. 05 H₂C H₂C base H base Enter base 88 Figure 24. Structure of DNA. The phosphate groups impart an overall negative charge to the molecule which means that it will migrate towards the positively charged pole when voltage is applied. However, since the amount of negative charge on a DNA molecule is proportional to its length, DNA molecules of different length have a similar q/f ratio and in theory migrate at a similar steady state velocity. This means of course that all DNA molecules regardless of their size would migrate at the same speed in electrophoresis. This should lead to the question of how electrophoresis can be useful to the researcher if all the DNA molecules behave the same. The answer to this can be found in the utilization of various types of gels. Agarose gel electrophoresis To separate DNA molecules of different lengths we need to perform the electrophoresis in a porous, solvent-filled mechanical support that is called a gel. Gels for molecular biology can be made from any material that forms a sieving matrix that has minimal interaction with the molecules that one is trying to separate. For DNA, the gels are usually made from agarose. Agarose is a linear polysaccharide that forms a mesh network in aqueous solution when it is melted in buffer and allowed to re-solidify. Agarose gels are typically used for the electrophoretic separation of large biomolecules such as nucleic acids and also of proteins with a mass greater than 250 kDa. The mesh network formed by solidification of molten agarose creates a gelatinous like substance that is filled with pores of varying sizes. One can yary the average pore size by changing the concentration of the agarose, with higher agarose concentrations resulting in a lower average pore size. When you apply a DNA sample to one end of a gel and activate the voltage, the negatively-charged DNA will begin to migrate towards the positively charged pole at the opposite end of the gel (see pictures below). While the solvent molecules can still easily pass KI Side view: Sample loaded into well Negative (-) Electrode Gel Electric field and direction of migration 89 Plastic gel box. buffer Positive (+) Electrode Figure 25. Photograph of DNA samples being loaded on an agarose gel (left), and Side view drawing of the electrophoresis chamber for an agarose gel (right). through the gel unimpeded, larger molecules like DNA can only migrate through the larger pores. Since there is a distribution of pore sizes, shorter DNA molecules will have a greater number of pores that they can migrate through compared to the larger DNA molecules. Since the smaller DNA molecules are able to more frequently find a pore that they can fit through, they migrate faster through the gel than the larger DNA molecules because they in effect travel a shorter distance for any linear distance along the axis of the gel in their journey towards the other end of the gel. We of course see this as them moving further along the gel compared to larger DNA molecules. This is what makes electrophoresis such a useful technique-it separates DNA molecules on the basis of their size. Once the electrophoresis is completed, one must have some way of identifying how far the DNA has moved-on-the-gel. Since DNA is not readily visible on the gel, chemicals that are easily detected and interact only with the DNA are used to "see" the DNA. There are many different dyes that can be used to visualize DNA, and the dye that you will be using in this lab is known by the trade name as Red SafeTM. This dye will be incorporated into the molten agarose solution before you come to the lab. To determine the size of the DNA molecules subjected to electrophoresis, one compares the migration distance of the DNA sample to the migration distance of a standard solution of DNA molecules of known sizes that were run on the same gel as the sample. Samples of DNA of known sizes designed to be run on gels are called DNA ladders.