Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/ca - PDF document

Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Restriction enzymes Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Sample Map Problem Experimental dataNumber of base pairs per banddIII / Here's our approach to solving this plasmid mapsample problemDetermine the number of base pairs (bp) in the wholeplasmid. Note that only one band results when the plasmid iscut by dIII. This is a very good clue that dIII has only onerecognition site, and that when cut, the plasmid ring opens intoa linear piece of DNA that is 50 bp long. This is confirmed bylooking at the other combinations of restriction enzymes cuttingthe plasmid into pieces that, each time, add up to 50 bp.Start drawing circles! (Note: Clock face references in thefollowing instructions indicate cut sites or where lines are to bedrawn.) The first circle should indicate one cut site for dIII at12:00. Label your circle with “dIII” (or “H” for short) above atick mark at 12:00 to indicate a single cut (a single digest) bythis restriction enzyme. See figure 1. This accounts for yourexperimental data from the single digest with Place the number 50 in the center of the circle to indicatethe total number of bp.Determine a scale for your circle. If the total number of bp goingaround the circle is 50, then 6:00 represents 25 bp, 3:00represents about 12–13 bp, and 9:00 represents about 37–38bp. See figure 2Draw another circle to indicate where the RI restriction sitesare located. The data indicates 3 different-sized bands resultwhen the plasmid is cut with just RI (a single digest).Start by placing a tick mark at 12:00, as in figure 2, toindicate the first RI cut site.Make another tick mark at about 7:00 to indicate a secondRI cut site 30 bp from the first site. See figure 3. Thisarrangement accounts for the 30 bp band in yourexperimental data. This leaves 20 bp between 7:00 and12:00. The other 2 data results, 15 and 5, add up to 20 bp.Go to approximately 11:00 or 7:00 (either option accounts forthe experimental data and is therefore acceptable becauseplasmid maps show locations) and make a thirdtick mark. See figure 4. For example, if you go toapproximately 11:00 (a distance of 5 bp), the remainingdistance to 7:00 represents 15 bp (for a total of 20 bp), whichaccounts for your experimental data.Fit the 2 circles together to account for the experimental datafrom your double digest (RI). We recommend the useof 3-D plasmid models (loops) to help students visualize thisprocess. You can make these loops out of inexpensive materialssuch as string, rubber tubing, paper strips, or Velcro strips.Mark one loop to indicate the dIII digest.Mark another loop to indicate the Place the dIII loop on top of the RI loop and align thedIII tick mark at about 2:30 on the RI loop, whichindicates a site 10 bp from the RI site at 12:00 and 20 bpfrom the RI site at 7:00. See figure 5 # of 5 50 6 7 30 8 9 10 20 11 12 13 14 15 15 16 17 18 19 10 20 21 22 23 24 25 5 26 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Bear in mind, it cannot be determined from the data if the dIII tick mark should be located 10 bp from the 12:00 siteor 10 bp from the 7:00 site. Both locations would account forthe experimental data. See figure 6Remember that these plasmid maps give relative positions.For more accuracy, you must perform additional digests.Have your students make a final “answer” plasmid map thatindicates each RI cut site, the dIII cut site, and accountsfor all experimental data.Emphasize to your students that their circles show relativelocations (loci) for the sites of the restriction enzymes. If theysimply turn their paper over, flip it upside down, or spin it 90°the picture changes, but the relative positions remain the same.Visit the Dolan DNA Learning Center at Cold SpringHarbor Laboratory's Web site for a look at a few plasmid mapsthat are used in some of our Carolina biotechnology kits.Carolina DNA mapping kitsRestriction Mapping of Plasmid DNAElectrophoresis of precut DNA with an analytical application. Students cast gels and perform electrophoresis of ready-to-load samples ofpredigested plasmid DNA. After staining with BLU™ stain, students determine the sizes of the plasmid DNA fragments in eachsample and use the data to deduce a restriction map of the plasmid.View Product Restriction Mapping of Lambda DNAThis kit provides data that allow students to assemble a map of cutting sites of I and 01091 on the genome of the bacterial viruslambda. This is accomplished by digesting lambda DNA with 2 restriction enzymes alone and in combination. The resulting restrictionfragments are subjected to electrophoresis in an agarose gel along with lambda/dIII size markers. The distances migrated by thedIII fragments vs. their base pair sizes are plotted on semilog graph paper. The distances migrated by the unknown fragmentsare then used to extrapolate their base pair sizes. Accounting for the appearance and disappearance of bands in single vs. multipledigests allows students to position the restriction sites relative to one another, resulting in a restriction map.View Product Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # 1Determine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental dataNumber of base pairs per bandEcoRI /# of 6 40 7 8 24 9 10 16 15 12 16 17 18 19 20 8 21 22 23 24 25 26 4 27 28 29 30 31 32 33 34 35 36 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # Determine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental dataNumber of base pairs per bandBamHI /# of 52 26 14 12 208 21 22 23 246 25 26 27 28 29 30 31 32 33 34 35 36 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # 3Determine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental data# of base pairs per bandEcoRI /EcoRI /EcoRI /BamHI /# of 1480.0 15 16 17 48.0 44.0 42.8 40.0 38.8 10.0 9.2 4.0 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # 4Determine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental dataNumber of base pairs per bandEcoRI EcoRI /# of 40 22 16 12 216 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # 5Determine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental dataNumber of base pairs per bandEcoRI EcoRI /EcoRI /BamHI /# of 87.2 79.6 72.2 64.6 22.6 15 7.6 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # 6Determine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental dataNumber of base pairs per bandEcoRI EcoRI /EcoRI /BamHI /# of 47.8 33.2 31.2 28.0 24.2 20.2 19.0 18.2 15.0 8.6 5.4 5.2 3.6 3.2 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # 7Determine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental dataNumber of base pairs per bandBamHI /# of 46.0 42.0 36.0 31.0 24.0 22.0 15.0 14.0 7.0 5.0 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # 8Determine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental dataNumber of base pairs per bandBamHI /# of 50.0 23.0 15.0 12.0 9.0 8.0 6.0 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # Determine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental dataNumber of base pairs per bandBamHI /# of 20.0 11.0 7.0 6.0 4.0 3.0 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # 10Determine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental data# of base pairs per bandBamHI /# of 40.0 16.0 12.0 9.0 6.0 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # 11Determine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental dataNumber of base pairs per band# of 20.0 11.0 8.0 6.0 3.0 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # 12Determine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental dataNumber of base pairs per bandBamHI /EcoRI /BamHI /Frag. Frag. # of 19.5 12.0 10.5 8.0 7.5 4.5 3.0 1.5 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # 13-ADetermine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental dataNumber of base pairs per band# of 30.0 628.0 726.0 8 22.0 18.0 14.0 12.0 10.0 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # 13-BDetermine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental dataNumber of base pairs per bandEcoRI /BamHI /BamHI /HindIII /EcoRI /EcoRI /# of 26.0 24.0 22.0 18.0 16.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0 Carolina: Plasmid Mapping Exercisesfile:///X|/IIS%20website/wwwroot/carolina_archive/biotech/plasmid_problems/plasmid_mapping_exercises_2008.htm Plasmid mapping: Exercise # 13-CDetermine the number of base pairs (bp) in the whole plasmid, and thendetermine a scale for your plasmid map. Visualizing the map as a clock face ishelpful. For example, if the total number of base pairs going around the map is50, then 6:00 represents 25 bp, 3:00 represents about 1213 bp, and 9:00 represents about 3738 bp.Experimental dataNumber of base pairs per bandBamHI /EcoRI /BamHI /EcoRI / PstIEcoRI /HindIII /BamHI /HindIII /BamHI /EcoRI /HindIII /# of 22.0 14.0 12.0 10.0 8.0 6.0 4.0 2.0