1.12: Restriction Digest with Gel Electrophorisis

Learning Objectives

Goals:

• Understand the function of restriction enzymes.
• Conduct analysis of DNA fragments by gel electrophoresis.

STUDENT LEARNING OUTCOMES:

Upon completion of this lab, students will be able to:

• Read a plasmid map to determine restriction sites and fragment sizes.
• Determine if restriction enzyme recognition sequences are palindromes.
• Predict the sizes of DNA fragments formed after a restriction digest.
• Compare gel electrophoresis bands to determine DNA sizes.

Introduction

Recombinant DNA technology is possible due to several tools useful for manipulating DNA molecules and transforming cells -- including plasmids, restriction enzymes and DNA ligase. This lab introduces you to plasmids and restriction enzymes, as well as the lab technique of gel electrophoresis. Later lab experiments will introduce you to the other tools of biotechnology.

Restriction enzymes (also called restriction endonucleases) are proteins made by many bacterial species, to defend against viral infections. Each restriction enzyme moves along a DNA molecule until it finds a specific recognition sequence in the DNA. The enzyme cuts the double-stranded DNA, resulting in DNA fragments. Over 3000 restriction enzymes that recognize short (4-8 bp) palindromic sequences have been discovered.

Figure 1. Recognition sequence for enzyme Hind III

Figure 1 shows the recognition sequence for restriction enzyme Hind III. Notice that the recognition sequence is a palindrome, and reads the same going forwards and backwards. The Hind III enzyme makes a staggered cut of the DNA, and produces fragments that have single stranded areas called “sticky ends”. Figure 2 shows the recognition sequence of two other restriction enzymes Sca 1 and Pst 1. Enzyme Pst 1 makes a staggered cut of the DNA at its recognition sequence. But restriction enzyme Sca I makes a blunt cut at its recognition sequence to generate DNA fragments with no sticky ends.

Figure 2: Restriction enzyme recognition sites.

Bacterial cells have all of their genes (genome) in a single circular chromosome. But bacterial cells can also carry non-essential pieces of DNA called plasmids. A plasmid is a small circular DNA that is able to replicate itself, and can carry a few genes from cell to cell. Scientists are able to design recombinant plasmids to carry specific genes into a target host cell.

Figure 3: Plasmid map of pUC19.

The genetic map of a plasmid “pUC19” is shown in Figure 3. The total size of the plasmid is 2686 bp. There is a Pst I recognition site at position 439, Hind III recognition site at position 447, and Sca I recognition site at 2179. If one restriction enzyme is used to cut pUC19 plasmid, what would be produced?

Determine what DNA fragments are produced when two restriction enzymes are used to cut pUC19 plasmid DNA.

Table 1. Predicted DNA Fragments from Restriction Digest of pUC19 Plasmid

Cut with Restriction enzymes	Sca I and Pst I	Sca I and Hind III	Pst I and Hind III
Resulting DNA fragment sizes

Part I: Restriction Digest

Agar is a polysaccharide derived from red algae. The agar powder is first dissolved in a boiling liquid, and then cooled to form a gelatinous solid matrix. As microbes cannot digest agar, this material is used commonly in laboratories to hold the nutrients that bacteria need.

Materials

• P-20 micropipette
• Box of disposable pipette tips
• Clean microfuge tubes
• Microfuge tube rack
• Permanent marker
• Waste container for used tips and microfuge tubes
• Microfuge tubes containing:
• pUC19 plasmid DNA
• pPUS2 plasmid DNA
• Pst 1 Restriction enzyme
• Sca 1 Restriction enzyme
• Restriction buffer
• Deionized water

Equipment

• Microcentrifuge
• 37^oC water bath (or dry bath or incubator)

Method

1. Use a Sharpie to label the top and side of 3 clean microfuge tubes A B C and your group name.
2. Follow the reagent table below and dispense the proper amounts of reagents to the labeled tubes. Use new tips for different reagents. Add reagents to the solution at bottom of tube. Always check that your pipet tip is empty after dispensing the reagent.

Table 2. Volumes of Reagents to Add to Each Tube

Tube	DNA	Pst 1 RE	Sca 1 RE	Restriction Buffer	Water
A	4 µL pPUS2	2 uL	none	4 uL	none
B	4 µL pUC19	2 uL	2 uL	2 uL	none
C	4 µL pUC19	2 uL	none	4 uL	none
D	4 µL pUC19	none	none	none	6 uL

3. Cap tubes tightly Place two tubes directly across from each other in the microcentrifuge.
4. Spin for five seconds to bring all the reagents to the bottom of each tube.
5. Place tubes into a floating rack in the 37°C water bath for at least one hour (but no more than two hours.)
6. After the incubation period is finished, you will analyze the contents by gel electrophoresis in Part IV.

Part II: Casting Agarose Gel

Agarose is a complex carbohydrate found in seaweed. If agarose is dissolved in a boiling liquid and then cooled, the solution converts into a solid gel matrix. The agarose solution will be poured into a casting tray to form the desired gel shape. A gel comb has teeth that is used to form the “wells” or holes for loading the samples. You will be prepare and cast a 1% agarose gel with electrophoresis buffer.

Materials

• Agarose powder
• Weigh boat
• Spatula
• Masking tape
• 100 Graduated cylinder
• 250 mL Erlenmeyer flask
• Electrophoresis Gel Casting tray
• Gel comb
• Deionized or distilled water
• 1X Electrophoresis buffer
• Heat-resistant silicone mitts or tongs
• Electronic or analytical balance
• Microwave

Method

Note

• Note: If you have concentrated electrophoresis buffer stock, you must dilute the stock to 1X working concentration before preparing agarose solutions or running gel electrophoresis.
• For 20X stock, combine 25 mL 20X stock with 475 mL deionized water to make 500 mL 1X buffer.
• For 50X stock, combine 10 mL 50X stock with 490 mL deionized water to make 500 mL 1X buffer.

1. Using the graduated cylinder, measure 100 mL of the 1X electrophoresis buffer.
2. Using an electronic scale, measure 1.0 g of agarose powder.
3. Pour some of the measured buffer into an 250 mL Erlenmeyer flask. Pour in the measured agarose powder. Pour some of the measured buffer into the agarose weigh boat, and pour into flask. Repeat until all agarose has been transferred to flask. Pour rest of buffer into flask.
4. Cover the opening of the flask with plastic wrap. Use a pipette tip to poke a small hole in the plastic wrap.
5. Place the covered flask in a microwave and heat on high. When you see bubbles form in the solution, pause the microwave, use oven mitts to gently swirl the flask a few times.
6. Continue microwaving the flask until the liquid starts to bubble again. Using oven mitts, hold the flask to the light and swirl the solution. Look carefully to check that there are no specks or swirls of agarose suspended in the liquid. If liquid is clear, then the agarose is dissolved. Wait five minutes for the agarose to cool. Note: Instructor will announce if you have a casting stand system and do not need to tape each tray.
7. Prepare the acrylic electrophoresis gel trays for casting. You may need to tape the two open ends of each tray. Be sure to press tape firmly along the entire edge of the tray with your fingernail. If using masking tape, you can see a difference in the tape translucence.

Figure 4. Casting tray with tape

8. Place a comb in each tray before adding the agarose solution.
9. When the agarose solution has cooled to the point that you can safely touch the bottom of the flask (~60°C; about five minutes), pour agarose solution into each casting tray, so that the solution covers about 2 mm of each comb. Note: Each Mini One gel requires 12.5 mL agarose solution, each casting tray holds two gels = 25 mL total.
10. Once the gels solidify (which will take around 30 minutes), pull the comb out of each gel. Pull it straight out without wiggling it back and forth; this will minimize damage to the front wall of the well.

Part III: Practice Pipetting

While you are waiting for the restriction digest to incubate, you can practice loading samples into a practice gel. As agarose gels are very easy to puncture through, it is important to have good technique for loading the samples. As the gel wells are small, only push the micropipette to the FIRST stop to dispense the sample. Purified DNA looks like water, so a colored dye is added to ensure that you can see the sample loading into the well. Glycerol, a viscous liquid, is added to the loading dye to ensure that the DNA sample will sink to the bottom of the well.

Figure 5. Pipetting a sample into the well of an agarose gel

Materials

• P-20 micropipette
• Box of disposable pipette tips
• Waste container for used tips
• Tube of colored dye/glycerol
• Agarose or polyurethane practice gel
• Agarose powder
• Weigh boat
• Spatula
• Masking tape

Method

1. Watch the video or instructor demonstration.
2. Submerge the practice gel with water or buffer.
3. Practice loading 5 µL and 10 µL colored dye into several wells of a practice gel. Do not change tips for this practice.
4. To steady your pipette hand, place your elbow on the table. You may also use your other hand to support and steady your pipette hand.

Part IV: Gel Electrophoresis

Gel electrophoresis is a technique to use electrical current to separate a mixture of molecules such as DNA, RNA, and proteins. The electrophoresis buffer contains ions to conduct electric current. As DNA molecules are negatively charged, they will migrate towards the positive electrode (red). The solidified agarose gel matrix will have pores of various sizes (similar to a sponge), so the size, shape and charge of the molecules can affect the rate of travel through the agarose gel. Smaller molecules move faster than the larger molecules.

Figure 6. Electrophoresis causes the molecules to separate by size in the porous gel

DNA can be visualized with various dyes. Scientists typically use ethidium bromide (either inside the agarose gel or as post-stain after the gel run). As ethidium bromide is mutagenic, we will not be using that in our class. Instead, we will use gel green stain, which is compatible with the blue LED transilluminators (eg. MiniOne). The alternative stain is gel red, which works with the uV transilluminators.

Materials

• Agarose powder
• Weigh boat
• Spatula
• Masking tape
• 100 Graduated cylinder
• 250 mL Erlenmeyer flask
• Electrophoresis Gel Casting tray
• Gel comb
• Deionized or distilled water
• 1X Electrophoresis buffer
• Heat-resistant silicone mitts or tongs
• Electronic or analytical balance
• Microwave

Method

Setting Up the DNA Samples

1. Find your tubes from the restriction digest (Part 1).
2. Add 2 µL of Gel green Loading dye into each of the sample tubes. Pipet up and down twice to mix the liquid.
3. Place tubes in a balanced configuration in a MicroCentrifuge and spin for five seconds.

Setting Up the Electrophoresis System

1. Watch a demo or assigned videos and follow instructions for placing the gel tray into the electrophoresis buffer tank.
2. Fill the buffer tank with 1X Electrophoresis buffer, ensuring that the entire gel is completely submerged. You want about 1 mm liquid layer above the gel, but not too much buffer as that can build up resistance.
3. Check that the gel is oriented with sample wells closest to the negative electrode (black). Check that the power cord can reach easily. Check that the gel box will not need to be moved for 30 minutes.
4. Draw and label in your notebook how the samples will be loaded in the gel. Check whether you will be sharing the gel with another group.
5. Using a new tip for each sample, load the DNA samples carefully into the gel wells.
6. After all the samples are loaded, place the cover over the electrophoresis box. [Note: Gel green is especially sensitive to light, so do not leave the Mini One light on during the electrophoresis].
7. Connect the electrical leads to the power supply. Connect both leads to the same channel, with the negative (-) cathode to cathode (black to black) and the positive (+) anode to anode (red to red). [Note: Mini One system must have orange hood in place to turn on].
8. Turn on the power supply and set the voltage to 130–135 V. [Note: Mini One systems do not have adjustable voltage].
9. After turning on the power on the gel boxes, look for bubbles forming on the negative electrode (to show electric current) and that dyes are moving toward the correct direction. If running the wrong way, wait until dyes are inside the agarose gel, then turn the gel 180o and restart the run..
10. Do not allow the loading dye to run off the gel. Be sure to turn off the power switch and unplug the electrodes from the power supply. Do this by grasping the electrode at the plastic plug, NOT the cord.
11. Carefully remove the cover from the gel box and pick up the gel tray. Slide the gel onto plastic wrap on top of the appropriate transilluminator. Take a photo.
12. Compare the sizes of the DNA ladder to the pUC19 fragments. The pPSU1 cut with Pst1 has fragments of 4100, 2000, 1000, 900, 800, 700, and 500. The pPSU2 cut with Pst I have sizes of 4100, 1500, 600, 500, 400, 300, 200, 100, 50.
13. What sizes are the pUC19 DNA fragments?

RESULTS

Figure 7: Restriction digests after electrophoresis

Study Questions

1. In nature, what is the function of restriction enzymes?
2. What is a palindrome? How does that relate to restriction enzymes?
3. Why do molecular biology research labs always have microwaves?
4. Why should you not ever eat in a molecular biology research lab?
5. How do you dispense samples into an agarose gel?