Download Directions and Questions for Lab 9 - San Diego Unified School District

Directions for AP Biology Lab
We will be making .8% agarose gels this time for our DNA digests. The purpose is to determine the basepair length of each DNA strand depending on which restriction enzyme was used.
1. Prepare a 0.8% agarose solution using the TBE buffer at 1x. Please make sure you have at least
30 mL of agarose to make your gel. We will want a gel that is thick enough to be handled.
2. Add stain to your gel
■■Adding Stain to Agarose
Adding gel and buffer stain to the gel and buffer allows observers to faintly see many of
the bands (DNA fragments) in the gel while the electrophoresis is running. It helps you to monitor the
progress of the electrophoresis.
You will still have to use the final stain to see all the bands more clearly. Decide, based on your results which
stain to use in the agarose.
The concentration of the stain added to the agarose/buffer is dependent on the voltage
used for electrophoresis. Add stain to the entire volume of the agarose. Just add the drops of stain to the
agarose, swirl to mix, and pour the gel immediately. We will be using 75 volts for our gels.
a. Use the TBE buffer at 1x to fill the apparatus and add your gel tray.
b. Load samples and make sure you to collect your data. You need to be certain which
sample is which.
3. Run the gel.
a. Making sure the cover is dry, place it onto the electrophoresis chamber. Wipe off any
spills on the apparatus before proceeding to the next step.
b. Connect the power supply. Making sure that the patch cords, as well as the female jacks
on the chamber, are completely dry, connect the red patch cord to the red terminal on
the power supply. Connect the black patch cord to the black terminal on the power
supply.
c. Once the power supply is connected to the patch cords, bubbles will form along the
platinum electrodes.
d. Observe the migration of the tracking dye down the gel toward the red (positive)
electrode.
e. Disconnect the power supply when the tracking dye has reached the end of the gel.
4. Stain the Gel
a. Slide the gel off the tray and into the stain.
b. Wearing protective gloves, pour approximately 100 mL of warm dilute stain into the
staining tray so the stain just covers the gel.
c. Let the gel stain for approximately 30-45 minutes.
d. Carefully decant the used stain. Make sure the gel remains flat and does not move up
against the corner. Decant the stain directly to a sink drain and flush with water.
e. Add distilled or tap water to the staining tray. To accelerate destaining, gently rock the
tray. Destain until bands are distinct, with little background color. This will take between
20 and 30 minutes, depending on the amount of agitation. Change the water several
times, or destain the gel, without changing the water, overnight.
f. View the gel against a light background, such as white paper, or on a light table. In the
Analysis section, roughly sketch the bands you see on the blank gel.
g. Gels can be stored in self-sealing plastic bags. For long-term storage, add several drops
of dilute stain to the bag to prevent the DNA bands from fading . If fading does occur,
the gel can be restained using the above procedure.
5. Analyze your gel.
a) Measure the distance of the DNA bands, in millimeters, from the bottom of the sample well
to the bottom of each DNA fragment on the marker. Measuring to the bottom of each
fragment band ensures consistency and accurate measurements. Do not measure the
migration distance of the largest fragment nearest the well; it will not be on the standard
curve and will skew results.
b) Record the measurements. Keep your data organized. This is a big part of this lab!
c) On semi-log graph paper, plot a standard curve for the DNA marker standard. Plot the
migration distance in millimeters on the X-axis, against the molecular size in base pairs (bp)
of each fragment. Draw the best-fit line to your points.
NOTE: When plotting on semi-log graph paper, the fragment size is expressed as a
logarithmic scale. Label the first series of lines 100 bp, 200 bp, 300 bp, etc. Then
label the second series of lines 1,000 bp, 2,000 bp, 3,000 bp, etc. The third series
would be 10,000 bp, 20,000 bp, etc.
d) Calculate the base pair size of each of the fragments by moving along the X-axis until you
have reached the distance traveled by the fragment. From that point, move upward until
you intersect the line of best fit on the graph.
e) Determine where that point is on the Y-axis and estimate the base pair value at that point.
Enter the values in the table.
6. Answer the questions
a) What are restriction enzymes? How do they work? What are recognition sites?
b) What is the source of restriction enzymes? What is their function in nature?
c) Describe the function of electricity and the agarose gel in electrophoresis.
d) How can a mutation that alters a recognition site be detected by gel electrophoresis?
e) Suppose you had an enzyme that recognized a sequence of six nucleotides. What are the
odds that this sequence would appear in a random chain of DNA?
f)
Given the answer to the above question, suppose you have a piece of human DNA that is
three billion base pairs in length. How many fragments will be generated by digesting the
DNA with the above enzyme?
g) In your lab, you ran your DNA samples on a 0.8% agarose gel. Would you get the same
results if you ran your samples on a higher percentage agarose gel? Why or why not?