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Making a Restriction Map

Making a Restriction Map . AP BIO. Step One. Look at the gel and determine the size fragments that are present in each lane. . BamHI 7 kb, 3 kb EcoRI 7 kb, 3 kb EcoRI + BamHI 3 kb, 2 kb. Step Two-Three. Draw a circle

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Making a Restriction Map

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  1. Making a Restriction Map AP BIO

  2. Step One • Look at the gel and determine the size fragments that are present in each lane. BamHI 7 kb, 3 kb EcoRI 7 kb, 3 kb EcoRI + BamHI 3 kb, 2 kb

  3. Step Two-Three • Draw a circle • Calculate the size (add up fragments from each of the restriction digests (each should add up to the same number) • Remember fragments of the same size will migrate the same distance and are not distinguishable if in the same lane BamHI 7 kb, 3 kb = 10 kb EcoRI 7 kb, 3 kb = 10 kb EcoRI + BamHI 3 kb, 2 kb = 5 kb ????? There must be two 3 kb fragments and two 2 kb fragments 3 + 2 + 3 + 2 = 10

  4. Step Four • Put in the first site • This is arbitrary---YOU have to decide where to put it • Hint: If there is one or more EcoRI sites, one of these is designated as the first site and is placed at 12 o’clock

  5. Step Five • Put the remaining EcoRI sites (or whatever sites of the enzyme you started with) • From the gel digest there are two fragments (7 kb and 3 kb). This means there are 2 sites.

  6. Step Six • Put in sites for a second enzyme, and distance these relative to the EcoRI sites as well as to each other • We know that the 2nd enzyme, BamHI, generates 2 fragments as well: 7 kb and 3 kb • So there must be 2 BamHI sites in the plasmid • We also know that the combination of BamHI and EcoRI generates fragments of 3 kb and 2 kb • Could any fragments from EcoRI remain in the double digest of BamHI? • YES! (3 kb is present in both) • Is it possible that both BamHI sites could be present in the remaining (7 kb) EcoRI fragment? • YES!

  7. Try This One: • A 8.9 kb circular plasmid is digested with three restriction enzymes, EcoRI, BamHI, and HindIII, individually and in combination, and the resulting fragment sizes are determined by means of electrophoresis. The results are as follows: EcoRI 8.9 kb BamHI 6 kb. 2.9 kb HindIII 8.9 kb EcoRI + BamHI 6 kb, 2.4 kb, 0.5 kb EcoRI + HindIII 7.4 kb, 1.5 kb BamHI + HindIII 5 kb, 2.9 kb, 1 kb EcoRI + BamHI + HindIII 5 kb, 2.4 kb, 1 kb, 0.5 kb

  8. Try This One: Answer There are two possible answers. Each map is a mirror image of each other.

  9. Need More Help? • https://www.youtube.com/watch?v=8FqMUF96cPE • https://www.youtube.com/watch?v=v2T8Y3-8674

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