Restriction of Lambda DNA

Advanced Preparation

• Agarose: Making up 0.8% agarose (notice this is agarose and NOT agar) can be done at any time, even weeks in advance. The agarose must be made using FRESH 1XTBE buffer NOT water. It does not have to be autoclaved only melted (clear not clouded). It can be stored in closed bottles at room temperature and melted by using a microwave oven or hot water bath. Be sure to loosen the bottle's cap before heating. Using the microwave is much simpler and quicker.
• Gel Preparation by Teacher: The volume of 1XTBE buffer needed to make 20 gels is approximately 800 ml. To pour the gels, you will need this volume of agarose times the number of classes doing this activity. If you decide to pour the gels (the procedure has the students pour them), you should pour the gels a few days in advance of the lab date. When taping the ends of a gel tray, be sure that the masking tape is pressed firmly along the edges of the tray. This will prevent leaks. Pouring the agarose should be done at approximately 60-65û C (Caution Hot). This temperature can be approximated by feel. If you can hold both hands around the bottle and not get a burning sensation, the agarose is at the correct temperature. However, for added protection, use hot gloves when pouring the gels. You should have as many gel trays prepared as you have agarose . Each gel requires 35-40 ml of agarose. Line the gel trays along the edge of a lab table and pour the agarose into one side. Pour until the agarose comes within 3/4 of an inch of the empty end. Stop pouring and allow the agarose to flow to the end of the tray. This exercise requires that the gels have at least 6 wells and that the Gel Comb be placed within 3/4 of an inch of the end of the tray. The gels can be stored inside zip lock bags at room temperature. Pour one or two extra so students who make major mistakes can be handed a new one to complete the exercise. If you have highly motivated or advanced students, gel pouring might be a special project for them to do as an after school exercise.
• Gel Preparation by Student: This exercise gives the instructions for students to pour their own gels. In order to follow this procedure, you must have some method for melting the agarose ahead of time ( a microwave is easiest) and a method for keeping the agarose at approximately 60û C (hot water bath) throughout the day. The number of water baths you use depends upon the amount of equipment available and how comfortable you are having them in the room. You can follow the above instructions for agarose preparation and determine how many bottles of agarose will be needed for the day. Gels can be stored in zip lock bags on the counter top for the student's use on the next day. Have students label their plastic bag and place their gel inside.
• Buffer: In addition to the 1XTBE buffer needed for the gels, you will need to make up approximately 500 ml for each electrophoresis chamber.Eight L of 1XTBE buffer should be enough for 16 chambers. You can reuse the 1XTBE buffer from the chambers so long as it is not badly contaminated by any student team. If contaminated, discard and replace with fresh 1XTBE buffer. If you have highly motivated or advanced students, buffer dilution might be a special project for them to do as an after school exercise.
• Enzyme: You will need to make up sets of enzyme for each student team by labeling microcentrifuge tubes (1.5 ml) as follows: B for BamH1; E for EcoR1; H for HindIII;and (-) for No enzyme. Transfer 15 µl of each enzyme into appropriately labeled microcentrifuge tubes. Since the exercise only requires 1 µl per reaction, each set contains enough enzyme to supply a team of 2 students for up to seven periods. Enzyme sets need to be kept FROZEN until ready for use. On the day of the exercise you will need to have ice containers at each lab station to keep the enzyme cold throughout the day. Suggestions would be crushed ice in Styrofoam containers or broken ice with water in empty pie tins or soup dishes. Pie tins and soup dishes work if the enzyme tubes are kept in the microcentrifuge tube racks so they don't turn on their side.
• Tip boxes: Be sure that all the micropipet tip boxes are filled. These tips need to be as clean as possible so filling the tip boxes should be done while wearing latex/vinyl gloves. This task is another one that can be done by your after school students or student service person.
• DNA Stain: You can dilute the DNA stain at any time. It can be stored at room temperature and can be reused many times. The volume needed to stain a gel is approximately 150 ml. For a lab of 16 stations, you will need 3 liters of stain.
• Preparation time: If the teacher chooses to pour gels for the class, the time required for 1 class set is 15-30 minutes. Time needed to dilute the DNA stain and transfer to staining trays is 20 minutes. Time needed to label microcentrifuge tubes and transfer enzymes is 30-45 minutes. Total estimated prep time 2-3 hours.

Introduction

This exercise is designed to introduce the student to the function of restriction enzymes and gain an appreciation of their use as molecular tools when working with DNA. The students will also see the connection between gel electrophoresis and imaging the result of the work of a restriction enzyme.

Student Objectives

• The student will identify restriction enzymes and their specificity.
• The student will determine the number of restriction sites on Lambda DNA.
• The student will visualize DNA pieces within a gel and effectively communicate this visualization.
• The student will estimate the size of each DNA piece produced by each enzyme.

Class Time Needed

Three 50-55 minute periods are needed to complete this exercise

1. On the first day of this exercise students should complete pouring a gel and setting up the restriction of Lambda DNA.
2. The second day is for loading the gel with cut Lambda DNA and separation by electrophoresis. Since loading the gel with cut DNA takes only 10-15 minutes, you need to have an additional activity ready to fill in the remainder of the period on this day.
3. The third day is for data analysis.

Materials

1. BamHI enzyme
2. 1XTBE buffer
3. EcoRI enzyme
4. Gel tray, comb, electrophoresis chamber
5. HindIII enzyme
6. 1/2 masking tape
7. Uncut Lambda DNA
8. Micropipet (1-20µl)
9. Microcentrifuge tube rack
10. Loading dye (10X)
11. Box of micropipet tips
12. Power supply
13. 0.8% Agarose
14. Waste container
15. Staining trays
16. DNA stain
17. Zip lock bags (sandwich size)
18. Permanent markers
19. Microcentrifuge tubes (1.5ml)
20. Spatula
21. Hot water baths
22. Ice baths
23. 2X Restriction buffer
24. Millimeter ruler
25. Semilog graph paper

Recipes for Consumables

1XTBE buffer: 100 ml of 10XTBE stock solution in 900 ml of distilled water.

0.8% agarose gels: 0.8 g of agarose powder melted in 99.2 ml of 1XTBE OR you can use the formula C1V1 = C2V2 to use any 2% agarose you may have left from other exercises. To make 100 ml of agarose at 0.8% from 2% agarose, use 40 ml of 2% agarose and dilute with 60 ml of 1XTBE. If you use this method, be sure to heat the 1XTBE so the melted agarose does not solidify when you pour the two solutions together.

DNA stain is made by diluting the stock solution 1 to 100. Dilute 10 ml of stock solution in 990 ml of distilled water.

Restriction buffer: The procedure is designed for this buffer to be at 2X concentration. If it is supplied at a higher concentration, use the formula C1V1 = C2V2 to dilute it to the 2X concentration. This MUST be done with DISTILLED WATER and JUST PRIOR to its use.

Procedure

Part A: Gel Preparation

Be sure that your students have taped the edge of their gel tray properly and placed the comb approximately 3/4 of an inch from one end of the tray.

They can then pour the gel as directed in the student procedure (similar to the teacher directions above) or you can have them transfer 40 ml of melted agarose into a 50 ml beaker and then pour this amount into the tray. It probably would be wise to have several sets of hot gloves in the room.

Part B: Loading the Gel

Loading the gel should not be a problem, however, you may wish to quickly review the micropipet and its use. Special attention to which stop for drawing up and which stop for expelling are probably the most important items to review. Lane 1 is usually defined as the outermost well of the gel on the black electrode side of the electrophoresis chamber. Once this well is loaded, the students can then load the wells in sequence according to the procedure.

Closing the electrophoresis chamber and connecting it to the power supply deserves special attention. Be sure that the lid goes on black lead wire to black electrode and red lead wire to red electrode. Also, be sure that the electrophoresis chamber is positioned where you want it on the table. Once the chamber has been connected to the power supply and the power has been turned on, the student is NOT to touch or handle the chamber. Check the lead wires from the lid of the chamber to be sure that they have been plugged into the correct receptacles of the power supply. Black to black and red to red and that the wires are plugged into receptacles that are next to each other. Once you have checked all of this at a lab station, you can turn on the power supply and start the experiment running. Set the power supply to a constant 100 volts and use this as the running voltage.

Separation of the Lambda DNA fragments will take at least an hour. This means that the electrophoresis process from one period will continue into the next. This poses a major equipment problem. You can either increase the size of the student teams to four, therefore needing 1/2 the number of gels, or arrange to borrow extra equipment from another teacher. Regardless of the decision you make, you will have to set up each lab station such that chambers from the previous class will be running while the current class is loading gels.

Staining gels can either be done in separate trays, one per lab station, or in a large, shallow pan big enough to hold the entire class set of gels. Pour enough stain into the tray to cover the gel completely. The gel needs to be taken off the gel tray in order to be placed in the staining tray. This should be done with a non- metallic spatula being careful not to damage it Special attention must be given to supporting the well portion of the gel since it will crack along the well line. Remove the gel from the stain in 30 minutes and place it in another tray of water (distilled is best but tap water works fine) for 90 minutes of destaining. Agitating the tray and changing the water 2 or 3 times helps the destaining process and should be done if you can manage it. After destaining, the gels can be stored in zip lock bags for several days. Place each team's gel in their labeled bag for future analysis. Although this stain is non-toxic, you should use latex/vinyl gloves while handling gels to keep your hands from being stained.

Disposal

• These gels can be placed directly into the garbage. It is probably best to put all the gels into a zip lock bag before placing in the garbage.
• Check with your math teachers to see if any of them intend to do the exercises that pertain to graphing the size of the DNA fragments versus their migration distances. You can save some of the better gels in zip lock bags so they can have them for their math classes.
• Be sure to save the 1XTBE from the electrophoresis chambers for future use.
• You can save all the DNA stain for future use so DON'T discard it.
• Save all left over enzyme and uncut Lambda DNA. These need to be frozen and stored in a NON-DEFROSTING freezer. If you have a freezer with an automatic defrost cycle, DNA and enzymes can be stored by keeping them in the styrofoam container they were shipped in from the supply company. They can be saved frozen for up to 1 year.