
Lab 9 Determining the Length of DNA Molecules Introduction A common and critical technique in the manipulation and analysis of DNA molecules is the accurate determination of molecular length, usually measured in nucleotide base pairs. Gel electrophoresis using an agarose matrix is the preferred method for separating DNA molecules longer than a few hundred base pairs. Polyacrylamide gels typically are used for smaller molecules. In both cases, the gel acts as a molecular sieve, separating molecules by size, with the smallest molecules migrating fastest and farthest. The resolving power of the gel is determined by pore size, which in turn is determined by the concentration of the gel (and for polyacrylamide, the extent of cross linking among polymers in the matrix). The physics of the movement of linear molecules through the pores in a gel is complicated, but generally migration distances can be plotted as a linear function of the logarithm of molecular length (for example, using semi log graph paper). Thus, the length of unknown DNA molecules can be determined if a standard curve of molecules of known lengths is developed and both sets of molecules are electrophoresed under identical conditions. The length of the unknown molecules are then calculated from the standard curve. (For long DNA molecules, length is generally reported in kilobases (kb), where 1 kb equals 1,000 base pairs. Short molecules are reported in base pairs (bp).) The figures below illustrate the principle.
In this experiment, you will determine the length of an unknown DNA fragment by comparing its electrophoretic migration to the migration of standard DNAs of known length. This process is an analytic technique rather than the testing of a hypothesis. The standard DNAs were generated by the enzymatic treatment of bacteriophage lambda DNA with the restriction endonuclease HindIII. The sizes of these standards are given below: Fragment Number Fragment Length (kb) 1 23.1 2 9.4 3 6.7 4 4.4 5 2.3 6 2.0 Procedures 1. Prepare a 0.8% (weight/volume) agarose gel using TBE buffer following the instructor’s directions. Approximately 75 mL of gel should be sufficient for the large gel size or 40 mL for the smaller gel. Use the comb appropriate for large well size. (Do not remove the comb until you are instructed to do so; you may damage the gel.) 2. Transfer your gel to the gel chamber in an orientation that places the sample wells at the cathode end (negative pole; black) of the electrophoresis chamber. Fill the chamber with buffer. DNA, an acid, is an anion at neutral pH and thus will migrate toward the anode. 3. Load 15 uL of each sample into the wells as indicated below. (See the Appendix for tips on using micropipettors.) Sample Well Number Sample 1 Dye mixture 2 Standard DNAs 3 Unknown DNA 4 Dye mixture
Note: Certain dyes can be used as standards to calibrate an agarose gel, and they allow a researcher to track the progress of an electrophoresis run. Dyes such as xylene cyanol, bromophenol blue, and orangeG are fairly accurate size indicators for small DNA fragments. The table below shows the approximate sizes of DNA with which these DNAs comigrate on a 1.2% gel. By running these dyes with your samples, you can roughly follow the progress of the electrophoresis run. Note: the dyes will move somewhat faster (like shorter DNA) on a 0.8% gel.
Dye Color BP Equivalent (at 1.2%) Xylene cyanol bluegreen 2800 Bromophenol blue purpleblue 250 OrangeG orange 70
4. Place the electrophoresis chamber lid in position, and with the power supply off, connect the cables from the chamber to the power supply, red to red (positive) and black to black (negative). Double check to make certain your gel is oriented properly. 5. Set the voltage at 150160 volts. Continue electrophoresis until the bromophenol blue in the dye mixture (and DNA samples) has migrated to within 5 mm of the anode (positive) end of the gel. 6. Remove the gel from the chamber and notice the relative distances the dyes have migrated from the sample wells. (The OrangeG will have already migrated off the gel.) 7. Carefully slide the gel off the casting tray and into the staining tray. Follow the directions for using "Instastain" 8. Measure (in millimeters) the migration distance of the DNA bands (standard DNAs and unknown) from the bottom of the sample wells to the middle of the bands representing the DNA.
Assignment 1. For this assignment, you will develop a “Results” section of a lab report. Results sections simply list data in a manner that is easy to interpret. Results do not include descriptions of how experimentsare conducted, which happens in a Methods section, and they do not interpret results, which is accomplished in the Discussion or Conclusion section of a report. Your results should consist of a table (typed) that lists your data in a clear and concise way, a graph (see step 2), and an application of your graph to analyzing the unknown data point (step 3). Make sure your table has a descriptive title at the top and that the columns and the rows are clearly labeled. Remember to include units where appropriate. 2. Develop a standard curve from your data using the attached semilog paper. Label the axes clearly. Plot the best straight line possible through the points. Neatness counts. (Each team member should do the graphing and analysis individually, although you may measure the gel together. In fact, it is good practice to check each other’s measurements.) Feel free to use a graphing program with a “best fit” plot if you desire, but remember that a log plot is needed to give a straight line. 3. From your curve (graph), determine the length of the unknown DNA fragment based on its migration distance.
Extra fun (credit): Your standard curve should be relatively accurate even if you draw the curve through the data points using only a ruler. For greater accuracy, a linear regression line can be plotted using a slope calculated from variance and covariance. With the known slope and yintercept, you then can calculate the size of the unknown rather than estimating it from the curve. If you enjoy stats, consider trying this option. 4. Food for Thought: The distance between two bases in a DNA double helix is 3.4A (angstroms, or 0.34 nm). What is the actual length of the unknown DNA that you detected with this technique? Show your calculations.
Appendix 1 Protocol for Using Micropipettes Note: DO NOT attempt to adjust the volume by turning the cylinder (control button) on top of the micropipettor until you are instructed to do so. These instruments are delicate and expensive. Instead, press gently down on the control button and notice that there are two “stop” points; the second is reached by gently pressing through the first stop. To withdraw and dispense liquids: 1. Attach a tip to the pipette without touching the tip with your hands. To do this, press the narrow end of the pipette directly into a tip as it sits in the box. (This avoids contaminating the tips with material from your fingers.) A gentle, even pressure is all that’s necessary to securely attach the tip. Lift the tip from the box with the pipette. 2. Press the control button down to the first stop and hold. 3. Hold the pipette vertically and immerse the tip approximately 3 mm into the liquid. 4. Let the control button glide back slowly and smoothly. 5. Slide the tip out of the liquid along the inside wall of the vessel. 6. To dispense the liquid, hold the tip at an angle against the inside wall of the receiving vessel (or gel well). 7. Press the control button slowly down to the first stop and wait approximately 2 seconds. 8. Press the control button down to the second stop to empty the tip completely. 9. Continue to hold down the control button and slowly slide the tip out along the inside wall of the vessel. 10. Let the control button glide back slowly. Eject the tip by pressing the control button to the final stop, which is beyond the second stop.
Appendix 2: Using SigmaPlot SigmaPlot In this example, you have just completed an experiment in which the pH of the culture media was varied and you recorded the number of cells at the various pHs. There are a couple of things that you can do with this data. First you might be just interested in generating a line graph of this data and labeling the axis. But you may also be interested in finding out if there is a relationship between the pH and the number of cells. You could do a regression analysis between the independent variable (the one that you have control over, this is normally plotted on the Xaxis of the graph), or the pH and the dependant variable. Note that you don’t have the 10 samples, but you can still get meaningful data. Here is some data: pH Number of Cells 7.0 2054 7.1 2194 7.2 2302 7.3 2440 7.4 2570 7.5 2707 7.6 2885
To do a regression plot and analysis to look for relationships you can open SigmaPlot and: 1. Put an arrow on column 1 and double click, then type in pH. This will label the column of data. Then go to column 2 and do the same thing for the data on cell number. 2. Once you have entered your data, there are a number of ways to generate a graph, here is one easy approach. Go the left of the screen and select the image of a scatter plot (upper left graph image). Then select a regressiontype plot (upper right graph image). 3. Next select the form of your data, in our case it is “XY pair”. 4. Select your independent variable, or the Xvariable, by clicking on the column that contains the data, in our case it is pH. Then select the data for the Yaxis by clicking on cell number. 5. Hit “finish”, a graph will be generated. You can edit the graph labels by double clicking on the name of the graph and axis to rename them as you wish. 6. Now go to the toolbars at the top of the page and select “statistics”, then regression. You can then highlight the results which should look like the data below. Because the linear relationship between distance traveled and fragment size in today’s lab is based on the log of the fragment size, we need to transform our know fragment lengths into log of fragment length. We can do this manually with a calculator or in SigmaPlot as follows: 1. Enter your data on fragment size (column 1) and distance traveled (column 2) 2. Open “Transforms” toolbar and select “Quick Transforms” 3. Click on column 3, the location that you want your transformed fragment lengths to go. This should put Col(3) in the box under “Equation” 4. Go to the right side of the equals sign and type in log(col(1)) where column 1 is the column where the fragment lengths are located. This takes column 1, logs it and places the log values in column 3. 5. Then follow the procedure above for making a scatter plot with a regression line and use the distance traveled (linear scale, column 2) and the log of the fragment lengths (column 3) for your X and Y values respectively.
6. If you are using a regression equation of the form y = mx + b to describe the regression line, remember that this is really in the form of log y = mx + b. Therefore, if you know x, and want to solve for y, you need to take the antilog of y to get the fragment size.
The data below is from the regression results. It gives the values for the straight line regression line that best fits the data. The equation is in the form of Y = b_{0} + b_{1}x. Where Y is the dependent variable (cell number), b_{0} is the intercept when X=0, b_{1} is the slope or the increase in Y per increase in X, and X is the independent variable. R is the correlation coefficient, while R^{2 }is called the coefficient of determination. Rvalues are measures of how well the calculated regression equation describes the data. Rvalues range from 1 to 0, with 1 being a perfect relationship, and 0 being no relationship. The column of function values just gives you predicted cell numbers at different pH’s using the regression equation that was generated from the data you generated. Below is a printout from the program: Plot 1 Order 1 Curve 1: Cell Number column 2: Coefficients: b[0] 7422.9642857141 b[1] 1352.5 r ² 0.9964249545
Function Values: x f(x) 7 2044.5357142857 7.012 2060.7657142857 7.024 2076.9957142857 7.036 2093.2257142857 