Dna Fingerprinting Lab

DNA FINGERPRINTING LAB

Pre-Lab Questions

1. What was used to cut/ cleave the DNA samples? Explain how this works.

2. What is the name of the part of the gel that will we load our DNA samples in to?

3. Name the process used to separate fragments of DNA.

4. Name a few factors which influence how quickly a molecule moves when placed in an electric field.

5. Do we expect the DNA to move toward the positive or negative pole? Explain your answer (i.e., WHY does it move this way?).

6. What type of gel are we using?

7. (a) Which DNA fragment will move fastest through the gel, one of 100 nucleotides (base pairs) or 1000 nucleotides (base pairs)?

(b) Which band would be found closest to the positive pole?

(c) Which band would be found closest to the negative pole?

8. What will need to be done to the gels so that we can actually see the DNA bands in the gel?

9. Explain the basics of how gel electrophoresis/ DNA Fingerprinting can be used to help solve criminal cases.

10. Why do you think scientists call this technique DNA fingerprinting? In what ways does this differ from actual fingerprint analysis?

Purpose

In this lab, to prepare and analyze a simulated DNA fingerprint, we will:

load an agarose gel with DNA samples pre-cut with the same restriction

enzyme,

conduct gel electrophoresis to spread out the mixture of DNA fragments,

analyze the resulting banding patterns or “DNA fingerprint,” and use it to solve

a crime.

Materials, per team

Gel electrophoresis box 1% Agarose Gel

1. Power supply pre-cut DNA samples

Casting tray and comb electrophoresis buffer (1xTBE)

P-20 micropipet and tips loading dye

1.5mL reaction tubes microcentrifuge

plastic staining tray Blue DNA Staining Solution

Methods

Part I: Preparing the Gel

1. Obtain a casting tray and comb.

2. Seal both ends of the casting tray with masking tape (write group name/ initials on tape).

3. Place the comb into the casting tray (if there are two possible slots, place it in the slot closest to the end of the tray, as opposed to the middle).

4. Obtainer container of hot liquid agarose. Once the solution has cooled to approximately 55C, pour the agarose evenly into the floor of the casting tray until it covers about 2/3 of the length of the comb’s teeth (about 30mL).

NOTE: If you get bubbles, gently try to pop them with a pipet tip.

5. Allow the gel to sit UNTOUCHED for about 5-10 minutes to solidify. It will turn from clear to cloudy as it solidifies.

6. Once the gel has solidified, gently pour a small amount of TBE buffer such that it forms a small layer of liquid on top of the gel to prevent dehydration of gel during storage.

***NOTE: Stopping point!!! If needed, you may preserve the gel in the refrigerator for at least a week. If using it immediately, go on to Step 6.

7. Carefully remove the comb from the gel. Pull straight up! Also remove the masking tape from the ends of the casting tray.

8. Carefully place the tray with the gel into the electrophoresis box such that the end with the wells is toward the side with the negative electrode (black). The DNA (negatively charged will run toward the positive electrode (red).

9. Since two electrophoresis boxes will share one power supply, move the two boxes close to their power supply so that later you will be able to connect both of them to the power supply without moving them any further.

10. Pour enough of the 1x TBE electrophoresis buffer into the electrophoresis box so that it just covers the gel. This buffer is “spiked” with a bit of blue stain to enhance the staining of the DNA.

Part II: Loading the Gel

1. Obtain DNA samples that have been pre-cut with the same restriction enzyme.

2. Give all of the tubes a quick spin (about 1 minute) in the microcentrifuge. Make sure the centrifuge is well-balanced by placing samples across from each other in a symmetrical fashion (if you only have 3 samples, prepare a fourth tube with a few drops of water in it so that it’s properly balanced).

3. Is your gel ready to load? It should be in the electrophoresis box, under buffer solution; the comb and tape should have been removed and the empty wells in the gel should be at the negative (black) end of the box.

4. Decide which sample will go in which well and draw a labeled diagram to use when analyzing results so you know which sample was in which lane (since you only have 3 samples to load, I suggest leaving wells in between samples empty).

5. Set the micropipet to 12 uL and carefully load the entire contents (12uL) from each tube into a separate well in the gel, using a micopipet with a fresh tip, in the following manner:

a. Lower the pipet tip under the surface of the buffer solution directly over the well, without actually touching the gel with the tip (which could cause tearing or puncturing of the well)

b. Gently press pipet plunger and slowly expel a sample into a well. Keep plunger depressed until the pipet is out of the gel box!

c. Change tips between samples!

Part III: Running the Gel

1. Slide the cover on the gel box such that the electrodes plug in (completely, black with black and red with red).

2. Turn the power on and adjust the knobs until it reads 100 V (you may need to go from low to high or vice versa).

3. Look for bubbles arising from the wires on the bottom edges of the gel box to confirm that current is running through the buffer solution (and producing gas bubbles as a result of electrolysis).

4. Wait 2-3 minutes and observe the gel to confirm that the tracking dye (blue) is moving toward the positive end of the gel.

5. It will take about an hour for the DNA to migrate across the gel. I will keep my eyes on them and shut off the current when the tracking dye gets close to the end of the gel (on the positive side); this will ensure a good spread of DNA fragments/ bands (which will not be visible until they are stained).

Part IV: Staining the Gel

1. Obtain a plastic staining tray and use masking tape to label it with your group’s initials.

2. Remove the casting tray (with the gel on it) from the electrophoresis box and CAREFULLY slide your gel off the casting tray and into its labeled staining tray.

3. Gently shake the stain solution well before pouring it into the tray so that it covers the entire gel.

4. Stain for 30-40 minutes. Pour the stain back into its bottle.

5. Add enough distilled water to cover the entire gel. Allow the gel to “destain” in the distilled water for about 30 minutes.

6. To store the gel: Place the gel in a ziplock bag with a small amount of distilled water in it (to keep it from drying out) and place it in the refrigerator. Make sure to label the bag with your group’s initials.

Part V: Observing the Gel

1. Carefully observe the banding patterns on the gel. Some bands may be hard to see (especially the smaller DNA fragments). Tips:

- if your gel is in a ziplock bag, it may help to remove the liquid from the bag

- sometimes it helps to place the gel on a white sheet of paper

- sometimes it helps to hold it up to the light and experiment with titling it in different angles toward the light to attempt to see as many bands as possible

2. Carefully draw a nice labeled diagram (to scale) of your gel (using information from your previous diagram to determine which samples are in which lanes).

3. Determine which suspect seems to be guilty, according to your DNA fingerprinting results.

Clean up!!!