Restriction Endonuclease Digestion of pUC18 Worksheet
Explain the gel electrophoresis pattern you obtained or the expected pattern if your results are unavailable (see image below). Use the steps below to guide your explanation. Include all results and answers to the steps or questions in your report.
Advice:
1. Think about what you did. Intact pUC18 DNA was digested and the resulting fragments were loaded into a lane in the gel and separated by their lengths.
2. Measure the standards yourself.
3. The standards are all LINEAR fragments of DNA. You can only compare other linear DNA fragments to these linear standards.
4. Use a piece of graph paper at least 10 by 10 centimeters.
5. ccc pUC18 means closed circular pUC18.
undigested
ccc pUC18 Hind III Hind III
Ase I Ase I Marker
distance length distance length distance length distance length length distance
(cm) (bp) (cm) (bp) (cm) (bp) (cm) (bp) (bp) (cm)
5000
4000
3000
2000
1500
1000
500
Expected Results
Use the idealized results shown above to complete these tasks and answer the questions listed. Please note, the undigested pUC18 lane has pUC18 DNA that was NOT treated with a restriction endonuclease. That DNA is in a supercoil form (ccc), a form quite different from a linear form. pUC18 is a circular DNA molecule. If you let a rubber band represent that circle, a supercoil is a tightly twisted rubber band. Furthermore, if you cut the rubber band once, all the twists relax and you are left with a “linear” piece of rubber band. Cutting the supercoiled pUC18 once also relaxes the DNA into a linear molecule.
The standards are all linear DNA fragments. You should only compare linears to linears. You cannot estimate supercoil size relative to linear sizes. Sizes of the fragments generated by digesting the DNA are estimated by comparing the migration distance of the unknown fragment to those of the standards.
1. Measure the migration distance of each DNA fragment. (The starting point is the lower edge of the gel well at the top of the gel diagram.) Use the table provided to organize your data and calculations.
2. Plot a standard curve by hand on graph paper. The x-axis should be the migration distance. The y-axis should be the length of the DNA. Make either a linear plot or a semi-log plot. Plot the DNA standards (markers) used for your gel. Connect the points, do not smooth out the entire curve. (Fragment lengths are best estimated through a direct comparison to fragments of a similar size. DNA migration through an agarose gel does not have a linear relationship to the length of the fragment (it’s a sigmoidal relationship). Do not try to form a linear relationship by drawing the best “fitting” line.)
3. Compare the migration distance of the pUC18 fragments to the standards and estimate their lengths. Again, use the table to organize your answers.
4. Without consulting the pUC18 map, try to determine the relative positions of the HindIII and AseI sites on pUC18. Explain your determination.
5. Of all the fragments generated in the gel diagram, which DNA fragment(s) have an intact ampicillin resistance marker? Please circle and label which fragment(s) in the gel diagram has the intact gene. (You may and will have to consult the map of pUC18 on the introduction page.)
6. Of all the fragments generated in the gel diagram, which DNA fragments have a piece of the pUC18 lacZ gene? Please draw a box around each fragment in the gel diagram that has a piece of the lacZ gene. (You may and will have to consult the map of pUC18 in the introductory material.)
7. What would happen if you cut human DNA with HindIII, isolated one DNA fragment, mixed it with the pUC18 cut with HindIII, and ligated the HindIII ends by reforming phosphodiester bonds? Draw the DNA fragments with the correct Hind III ends. (Hint, what is the structure of each DNA fragment end and what is the DNA sequence?)
Use the space below for your answers. (Questions 1 and 3 use the table provided. Question 2. use graph paper. Questions 5. and 6., use the idealized gel diagram.)