Restriction Enzymes and Lambda DNA Computer 6B Restriction enzymes have become an indispensable tool of molecular researchers over the past fifty years. This unique group of enzymes function as molecular scissors when applied to nucleic acids, in this case DNA. Forms of this special class of protein have been isolated from several species of bacteria and are employed to make predictable, precise cuts in experimental DNA samples. Currently, over two hundred different restriction enzymes are available to researchers, each keying on a unique nucleotide recognition sequence. The mode of action of a restriction enzyme is to attach and scan a strand of DNA looking for the presence of a specific nucleotide sequence. When found, the DNA is cleaved at two opposite positions of the recognition site on the sugar-phosphate backbone. During this lab activity, prepared samples of bacteriophage lambda DNA (λ DNA) are used to perform agarose gel electrophoresis. The samples result from λ DNA being digested with different restriction enzymes. The individual digests of this bacteriophage, a 48,502 base pair linear DNA segment, use two common restriction enzymes; EcoRI and HindIII. One of samples is digested by EcoRI while another is cut by HindIII. There is a sample that is formed from a dual digest that has both enzymes acting simultaneously on λ DNA. As a control, an uncut form of λ DNA is also used. The technique of agarose gel electrophoresis relies on an electric field being applied to a charged gel matrix containing polar molecules. The response of these molecules to the electric field induces them to migrate through the gel to the pole with an opposite charge. The rate of molecular movement in a gel is determined by the charge, shape, structure and weight of the molecule being studied. Negatively charged phosphate groups are present in DNA nucleotides causing the molecule to migrate toward the positive end of the gel chamber. DNA fragments maintain the same charge, shape, and structure, so base pair number differentiates the molecules migration through the gel. During this exercise, gel electrophoresis will be performed using the E-Gel Pre-Cast Agarose Electrophoresis System with SYBR Safe stain. The Blue Digital Bioimaging System and Logger Pro software will also be used to capture and analyze a digital photograph of your electrophoresis results. OBJECTIVES In this experiment, you will copy Perform agarose gel electrophoresis with the E-Gel System using four different samples of λ DNA. Document and examine gel results with the Blue Digital Bioimaging System. Use Logger Pro to construct a standard curve and determine the base pair values from the gel. Evaluation Advanced Biology with Vernier 6B - 1
Computer 6B MATERIALS computer λ DNA samples Logger Pro λ DNA uncut Blue Digital Bioimaging System λ DNA EcoRI digest ProScope HR λ DNA HindIII digest BlueView Transilluminator λ DNA EcoRI/HindIII digest 1 10X lens 200 µl sterile water - ddh 2 O hood lab mat stand Nitrile gloves E-Gel Power Base & AC adapter microtube rack 1.2% agarose E-Gel (SYBR Safe Stain) ruler 2 20 µl pipettor waste container 2 20 µl sterile pipettor tips PROCEDURE Part I Perform Gel Electrophoresis 1. Prepare the E-Gel and the E-Gel Power Base. a. Clean the lab table surface, wash your hands, glove, set the lab mat, and review lab safety procedures. b. Power the E-Gel Power Base with the AC adapter. c. Remove the E-Gel from its packet and position it in the power base starting with the right edge so the gel electrodes make contact with the power base electrodes. Press the E-Gel down to lock it in place. A red light should go on and remain on at the top of the power base. d. (Pre-Run) Press and hold the 30-minute button on the power base until you hear the double beep and the green light starts blinking. When you release the button, a required two-minute warm-up cycle will begin. When the warm-up cycle is complete, the power base will beep repeatedly. Press and release the same button to deactivate this warning. A steady red light will appear. e. The set of microtubes includes uncut λ DNA, λ DNA- EcoRI digest, λ DNA-HindIII digest, λ DNA- EcoRI/HindIII digest, and ddh 2 O. Tap down each microtube. This action maximizes the amount of solution at the bottom of each tube. f. Remove the clear plastic comb from the top of the E-Gel and place it in the waste container. 2. Load the E-Gel. a. Twelve lanes are available in this E-Gel. Your instructor will suggest a loading sequence for the λ DNA set. Write down your lane assignment information in Table 1. b. Adjust the pipettor volume to 20 µl. Place a sterile tip on the pipettor, draw up the first sample, load it in its designated well, and eject the tip into a waste container. Repeat this step with each of the remaining three samples using a clean tip for each sample. Note: Each well of the E-Gel requires a total volume of 20 µl. If there are blank wells, fill them each with 20 µl of sterile water before running the gel. 6B - 2 Advanced Biology with Vernier
Restriction Enzymes and Lambda DNA 3. Run the E-Gel. a. Once all the wells are loaded, press the 30-minute button on the E-Gel power base to start the electrophoresis run. The red light on the E-Gel power base should turn green to indicate the run has begun. b. While the E-Gel is running, clean up your work area by returning your materials to the designated storage places. Dispose of nitrile gloves and wash your hands. c. At the conclusion of the thirty-minute gel run, the E-Gel power base will beep repeatedly and the light will flash red. Press one of the buttons to stop the beeping, disconnect the power cable from the power base, and remove the E-Gel. 4. Use a ruler to measure, in millimeters, the distance across the top of E-Gel from the start of the first well to the end of the last well. This distance will be used in Step 12. Record this value in Table 2. Part II Photodocumentation of Results 5. Start Logger Pro and choose New from the File menu. 6. Prepare the E-Gel and the BlueView Transilluminator. a. Transfer the E-Gel to the central portion of the blue platform of the BlueView Transilluminator. The top region of the E-Gel should be next to the hinge of the orange lid. b. Connect the BlueView Transilluminator to AC power and turn it on. 7. Position the ProScope. a. Connect the 1 10X lens to the ProScope. b. Connect the ProScope to the USB port. c. Mount the ProScope to the stand and position the stand next to the transilluminator, opposite the side with the hinge. d. Level the ProScope so that its lens is parallel to the surface of the transilluminator. 8. Prepare Logger Pro for use. a. Choose Gel Analysis Take Photo from the Insert menu. b. Orient and focus the ProScope so both the bands and lane numbers are clear and sharp. Note: Adjusting brightness to a lower value under camera settings is often helpful. Figure 1 9. Place the Imaging Hood over the ProScope and the BlueView Transilluminator. Reach through the flap of the hood to make final adjustments for best position, focus, and resolution. 10. Once satisfied with the image, click. The screen should now resemble Figure 1. Advanced Biology with Vernier 6B - 3
Computer 6B Part III Gel Analysis The buttons along the right side of the gel photograph are used during gel analysis. The first four are the primary Gel Analysis tools. Text above the photograph serves as a reminder of the next step in the analysis. 11. Indicate the position of the wells on the photograph. a. Click Set Origin,. b. Click the photograph just to the left of the first well. A yellow coordinate system will appear on the photograph. c. Position the x-axis directly along the bottom edge of the wells. You can move the origin by clicking either axis and dragging it to the desired location. The axis can be rotated by clicking the round handle on the x-axis. 12. Convert the units of distance measured from pixel count into millimeters or centimeters. a. Click Set Scale,. b. Click and drag between the start of the first well and the end of the last well. c. Enter the distance value from Table 2, including units. Click. 13. Identify the bands and base pair values of the standard ladder using the λ DNA/EcoRI digest lane as the standard ladder. a. Click Set Standard Ladder,. b. Click the leading edge of the first band in the λ DNA/EcoRI digest lane. c. Enter the number of base pairs for this band using the values in Table 3. Click. d. Click the next band in this lane and enter the base pair value. Click. e. Repeat this process for each visible band of the standard ladder. Logger Pro will automatically create a standard curve on the graph. 14. Identity the experimental bands in the remaining lanes. Logger Pro will plot bands, record distance migrated and calculate the respective number of base pairs. a. Click Add Lane,, and choose Add Lane. b. Click the leading edge of the first band in the first experimental lane. Notice that when you click, three things happen: a marker with a distinct shape and color is placed on the photograph, a matching marker is placed on the standard curve of the graph, and the distance and number of Figure 2 base pairs are added to the data table (see Figure 2). c. Click the leading edge of the next band in this lane. d. Continue this process for each visible band in the experimental lane. 15. Repeat Step 14 for each remaining experimental lane. 16. Record the base pair values for the experimental lanes in Table 4. Not all cells will be filled. 6B - 4 Advanced Biology with Vernier
Restriction Enzymes and Lambda DNA 17. (optional) Print the results of the E-Gel analysis. DATA Table 1 Lane assignments Lane Volume λ DNA (µl) λ DNA form used Table 2 E-Gel scaling sistance Distance across wells mm Table 3 Standard ladder values or λ DNA EcoRI digest Band Base pairs 1 21,226 2 7,421 3 5,904 4 5,643 5 4,878 6 3,530 Advanced Biology with Vernier 6B - 5
Computer 6B Table 4 Results λ DNA HindIII λ DNA EcoRI/HindIII λ DNA Band Base pairs Band Base pairs Band Base pairs 1 1 1 2 2 2 3 3 3 4 4 4 5 5 5 6 6 6 7 7 7 8 8 8 9 9 9 10 10 10 QUESTIONS 1. Which restriction enzyme produced the most DNA fragments during the digest of λ DNA? 2. The gel used in this activity was 1.2% agarose. If the concentration of agarose were 2%, what effect would this have on the migration of DNA fragments? What effect would a 0.8% agarose gel have on this activity? What benefit would exist in using a higher concentration agarose gel? 3. DNA molecules consist of two complimentary chains of nucleotides arranged in an antiparallel fashion to form a helical structure. This double stranded molecule contains paired nucleotides following Chargoff s Rule. Each nucleotide position along a segment of DNA can be one of four forms; adenine, thymine, guanine, or cytosine. Considering the six base pair recognition sequence for the restriction enzyme EcoRI, how often would you expect this sequence to appear? 5 GAATTC 3 3 CTTAAG 5 Recognition sequence for Eco RI Assuming your research project deals with an organism whose genome contained 12 million base pairs. You have isolated and cleaned up a sample of the organisms DNA and want to make a collection of smaller segments that you will use for the next stage of your research. How many segments would you expect to result from a digest with EcoRI? 6B - 6 Advanced Biology with Vernier
Restriction Enzymes and Lambda DNA 4. As observed from the results of your gel run, smaller fragments appear further from the well than larger segments. Explain why the shorter DNA segments migrate furthest during agarose gel electrophoresis. 5. Why is a semi-logarithmic graph used when creating a standard curve to determine base pair lengths of experimental DNA segments? 6. What affect would reducing the voltage to your gel by twenty percent have on the observed results? 7. Restriction enzymes make one of two types of cuts in DNA being digested. Some of these enzymes produce sticky ends at the cut site while others produce blunt cuts. What are sticky ends and what makes them important to recombinant DNA studies? 8. During a restriction digest using HindIII on DNA isolated from an insect, gel electrophoresis was performed using a 1.0 % agarose gel. The results showed several bands with the same light intensity. One band, however, was several times brighter than the others. Further investigation in the literature revealed this brightness was due to a triplet of closely related DNA segments. What could you do to better resolve these three bands in your gel? Considering the activity just performed, how might you expand it to resolve a base pair count for each of the bands? 9. Restriction enzymes can protect bacteria from many viral infections by attacking foreign DNA and cutting it up into useless segments. What would stop a restriction enzyme from digesting the DNA of the host bacteria that created it? 10. A process called restriction fragment length polymorphism (RFLP) has been used to initially assess relatedness of different species. Explain how this process works. Discuss its limitations. EXTENSION 1. Restriction enzymes can be used with smaller DNA vectors, specifically plasmids, to give much more controlled results. Using plasmid X, devise a map of this circular structure based on the following restriction digest results. The uncut plasmid is known to consist of 4361 base pairs. Three restriction enzymes were used to characterize the plasmid through a series of dual digests; enzymes A, B, and C. Results were run on an agarose gel and the banding patterns were analyzed. Digest with enzymes A and B yielded two segments whose lengths were 377 and 3984 base pairs, respectively. The second digest using enzymes A and C produced a segment 748 base pairs long and another of 3613 base pairs. The final digest using enzymes B and C resulted in two segments, one 1125 base pairs and the other with 3236 base pairs. 2. Using commercially available plasmids and restriction enzymes, e.g., puc 19 and BsaI, EcoRI, and HindIII, digest a plasmid with three separate dual digests. Use a 1.2% agarose gel to separate the digest results. A low range DNA ladder will need to be used as a standard and the results can be analyzed with Gel Analysis. From your results, reconstruct the plasmid and its restriction sites. Advanced Biology with Vernier 6B - 7
Vernier Lab Safety Instructions Disclaimer THIS IS AN EVALUATION COPY OF THE VERNIER STUDENT LAB. This copy does not include: Safety information Essential instructor background information Directions for preparing solutions Important tips for successfully doing these labs The complete Advanced Biology with Vernier lab manual includes 18 labs and essential teacher information. The full lab book is available for purchase at: http://www.vernier.com/cmat/bioa.html Vernier Software & Technology 13979 S.W. Millikan Way Beaverton, OR 97005-2886 Toll Free (888) 837-6437 (503) 277-2299 FAX (503) 277-2440 info@vernier.com www.vernier.com