RESTRICTION ENZYME AP LAB
Materials:
Electrophoresis Gel
DNA Samples
masking tape
gel tray
micropipette & tips
buffer
staining chamber
de-staining chamber
methylene blue stain strips
spatula
distilled water
light box
Procedure:
Setting Up the DNA Electrophoresis Gel:
1. Use the spatula to carefully lift your gel from the buffer. Do not allow gel to remain exposed to open air.
2. Place the gel in the tray with the wells at the end of the tray labeled with the negative sign. Very carefully, carry the gel back to your table
taking care to protect the open ends of the tray.
3. Submerge the plastic tray containing the gel in the set-up chamber. Make sure the gel is completely covered with buffer.
4. Inoculate each well using the following procedure.
 Place an unused disposable pipette tip on the micropipette.
 Prepare to load the micropipette by pushing down the plunger completely, inserting the micropipette into the sample, releasing the
plunger slowly in order to withdraw the sample into the pipette.
 Carefully lower the micropipette through the buffer into the first well of the electrophoresis gel. Load the gel by pushing down on the
plunger carefully, releasing the contents of the pipette into the small well completely without touching the gel itself.
 Withdraw the pipette. Discard the tip.
5. Inoculate the wells in the following order:
Well 1 = Marker DNA (lambda cut with HindIII/EcoRI)
Well 2 = PCR reaction performed with DNA with known CF mutation
Well 3 = PCR reaction performed with DNA with known wild-type sequence
Well 4 = PCR reaction performed with Child 2’s DNA
Well 5 = PCR reaction performed with Child 1’s DNA
Well 6 = No DNA template (control)
Running DNA Electrophoresis:
1. Load the gels into the specified chamber. Make sure the wells are closest to the negative terminal of the chamber.
2. After all gels are loaded in the electrophoresis chamber, slide the cover on. Make sure the electrode terminals are making good contact.
3. Set the power source to 75 Volts.
4. Plug in the power source, turn it on, and look for tiny bubbles forming on the sides of the chamber.
5. Allow the DNA to migrate for approximately 45 minutes or until the tracking dye has moved 3.5 to 4 cm from the wells.
6. After electrophoresis is completed, turn off the power, unplug the power source, disconnect the leads, & remove the cover.
Staining the Electrophoresis Gel:
1. Use masking tape to label the staining chamber with your table number.
2. Use the spatula to carefully remove the gel from the electrophoresis chamber.
3. Gently slide electrophoresis gel into staining chamber.
4. Pour enough Carolina BLU Final stain into the staining tray to just cover the gel.
5. Keep the gel in the stain for 20 minutes.
6. Pour the Carolina BLU stain back into the beaker on the side counter. Rinse the gel with distilled water, and then fill the tray with just enough
distilled water to submerge the gel.
7. Put the gel in the refrigerator overnight to de-stain.
8. Follow instructions on the back under De-staining and data collection.
Data Table Construction
1. Construct a Data Table on the 2nd page of your lab book using the following as a guide:
III – Results
DNA Fragment Migration Distance
Lambda Marker
Hind III/EcoRI
Distance
BP
Traveled Length
(cm)
xxx
21,226
5148
4973
4268
3530
2027
1904
1584
1375
947
831
564
125
Sample from Child 1
Distance
Traveled
(cm)
Interpolated
BP Length
Sample from Child 2
Distance
Traveled
(cm)
Interpolated
BP Length
Mutant DNA
Distance
Traveled
(cm)
Interpolated
BP Length
Wild Type DNA
Distance
Traveled
(cm)
Interpolated
BP Length
De-staining and Data Collection
1. After overnight incubation,
 Pour the water from your de-staining chamber into the sink.
 Fill your de-staining chamber with clean distilled water and de-stain for 5 minutes.
 Remove the gel. Blot gently.
 Look for stained bands of DNA. If needed, repeat de-staining procedure with clean distilled water.
2. Once de-staining is complete, obtain a small piece of transparent plastic wrap and a Sharpie. Wrap the plastic wrap completely around the
gel. Take care to smooth out any wrinkles.
3. Place the gel on the light box and mark each stained DNA band with a Sharpie. In addition, trace an outline of the gel and the sample wells.
4. Measure and record the distance traveled by each of the Standard DNA fragments.
 Measure from the lower edge of the sample well to the lower end of each band.
 Complete and record all measurements in cm.
 Do not measure the largest fragment, representing 21,226 bp as this will skew the line on your graph.
 Record measurements in Data Table.
5. Repeat the same process to measure the distance traveled by each of the bands in the lanes inoculated with DNA from Child 1, Child 2,
Mutant DNA, and Wild-Type DNA. Please note: There will not be as many bands as compared to the Lambda Marker so you will have
extra rows in the Data Table.
6. Compare the DNA fingerprints of child 1 and 2 to the mutant and wild type DNA. Determine if the children match the mutant or wild type DNA.
You will use this knowledge to answer analysis questions so make sure you make a determination before throwing away your gel.
Gel Analysis
1. Use the semi-log graph paper provided to construct the graph.
 Title the graph.
 Plot the data points for the Lambda Marker DNA Fragment.
 Draw a line of best fit. **Keep in mind that the point for the 21,226-bp or the 564-bp band may be an outlier. If this is the case, these
points (but not any of the other points) may be excluded from the line. This best-fit line is the standard curve.
2. To determine the number of base pairs represented by each fragment of DNA from Child 1,
 Locate on the X-axis the distance traveled of the first fragment in the lane with the sample generated from Child 1. Using a ruler,
draw a vertical line from this point to its intersection with the standard curve (the best-fit line you just drew). Extend a horizontal line
from the point where your vertical line intersects the standard curve to the y-axis. The point where the horizontal line intersects the
y-axis indicates the interpolated base-pair size of the DNA fragment.
 Repeat the procedure above for the DNA from Child 2, Mutant, and Wild-Type and place the answers under the interpolated bp in
the Data Table.