Experimental data. Sample Map Problem. Number of base pairs per band EcoRI HindIII / EcoRI

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1 Sample Map Problem / Here's our approach to solving this plasmid map sample problem. Determine the number of base pairs (bp) in the whole plasmid. Note that only one band results when the plasmid is cut by. This is a very good clue that has only one recognition site, and that when cut, the plasmid ring opens into a linear piece of DNA that is bp long. This is confirmed by looking at the other combinations of restriction enzymes cutting the plasmid into pieces that, each time, add up to bp.. Start drawing circles! (Note: Clock face references in the following instructions indicate cut sites or where lines are to be drawn.) The first circle should indicate one cut site for at :. Label your circle with (or H for short) above a tick mark at : to indicate a single cut (a single digest) by this restriction enzyme. See figure. This accounts for your experimental data from the single digest with. a. Place the number in the center of the circle to indicate the total number of bp.. Determine a scale for your circle. If the total number of bp going around the circle is, then : represents bp, : represents about bp, and : represents about bp. See figure.. Draw another circle to indicate where the restriction sites are located. The data indicates different-sized bands result when the plasmid is cut with just (a single digest). a. Start by placing a tick mark at :, as in figure, to indicate the first cut site. b. Make another tick mark at about : to indicate a second cut site bp from the first site. See figure. This arrangement accounts for the bp band in your experimental data. This leaves bp between : and :. The other data results, and, add up to bp. c. Go to approximately : or : (either option accounts for the experimental data and is therefore acceptable because plasmid maps show relative locations) and make a third tick mark. See figure. For example, if you go to approximately : (a distance of bp), the remaining distance to : represents bp (for a total of bp), which accounts for your experimental data.. Fit the circles together to account for the experimental data from your double digest (/). We recommend the use of -D plasmid models (loops) to help students visualize this process. You can make these loops out of inexpensive materials such as string, rubber tubing, paper strips, or Velcro strips. a. Mark one loop to indicate the digest. b, Mark another loop to indicate the digests.. Place the loop on top of the loop and align the tick mark at about : on the loop, which indicates a site bp from the site at : and bp from the site at :. See figure. Blank Blank Blank Blank Blank Base mm Pairs

2 a. Bear in mind, it cannot be determined from the data if the tick mark should be located bp from the : site or bp from the : site. Both locations would account for the experimental data. See figure. b. Remember that these plasmid maps give relative positions. For more accuracy, you must perform additional digests.

3 Plasmid mapping: Exercise #, then : represents bp, : represents about bp, and :.... /.... Blank Blank Blank Blank Blank Base mm Pairs

4 Plasmid mapping: Exercise #, then : represents bp, : represents about bp, and :..... /.... Blank Blank Blank Blank Blank base

5 Plasmid mapping: Exercise #, then : represents bp, : represents about bp, and : base pairs per band / / / / Size Standard Blank Standard I Blank Blank Blank base

6 Plasmid mapping: Exercise #, then : represents bp, : represents about bp, and :... /... Blank Blank Blank Blank Blank base

7 Plasmid mapping: Exercise #, then : represents bp, : represents about bp, and :... /.. /.. /.. Blank Blank base......

8 Plasmid mapping: Exercise #, then : represents bp, : represents about bp, and : /.... /.... /.... Blank Blank base

9 Plasmid mapping: Exercise #, then : represents bp, : represents about bp, and : / Blank Blank Blank Blank Blank mm base pairs

10 Plasmid mapping: Exercise #, then : represents bp, : represents about bp, and :..... /..... Blank Blank Blank Blank Blank base

11 Plasmid mapping: Exercise #, then : represents bp, : represents about bp, and :.... /.... Blank Blank Blank Blank Blank base......

12 Plasmid mapping: Exercise #, then : represents bp, : represents about bp, and : base pairs per band.... /... Blank Blank Blank Blank Blank base.....

13 Plasmid mapping: Exercise #, then : represents bp, : represents about bp, and :.... /... Blank Blank Blank Blank Blank base.....

14 Plasmid mapping: Exercise #, then : represents bp, : represents about bp, and :.. /.. / /.... (Fragment.. A) (Fragment.. B) Blank Frag. A Frag. B Blank base

15 Plasmid mapping: Exercise # -A, then : represents bp, : represents about bp, and : Blank Blank Blank Blank base

16 Plasmid mapping: Exercise # -B, then : represents bp, : represents about bp, and : /.... /.... /.... /... /... /.... Blank Blank base

17 Plasmid mapping: Exercise # -C, then : represents bp, : represents about bp, and : / /.... /.... / / /.... / /..... / /.... / Blank Blank Blank base pairs mm