Using the Digital Micropipet

Advanced Preparation

• Agar: Making up 2% agar can be done at any time, even weeks in advance. 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. The microwave is much simpler and quicker.
• Gels: You should pour the agar gels a few days in advance of the lab date. They can be stored inside zip lock bags at room temperature or in the refrigerator. Pour a few extra so students who make major mistakes can be handed a new one to complete the exercise. Tape the ends of the gel tray, being sure that the masking tape is pressed firmly along the edges of the tray. This will prevent leaks. Pouring the agar should be done at approximately 60-65û C (Caution Hot). This temperature can be determined by feel. If you can hold both hands around the bottle and not get a burning sensation, the agar 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 agar. Each gel requires 35-40 ml of agarose. Line the gel trays along the edge of a lab table and pour the agar into one side. Pour until the agar comes within 3/4 of an inch of the opposite end. Stop pouring and allow the agar to flow to the end of the tray. Don't forget, this exercise requires that the gels have at least 12 wells so be sure to pour them with 2 combs. The combs can be placed in any position within the tray but avoid placing them at the very ends. You can make satisfactory gels for this exercise by pouring into the bottom half of a disposable petri dish rather than using gel trays. You may want to do this if the number of gel trays is limited or to save the time of having to tape the trays. If you have highly motivated or advanced students, this preparation might be a special project for them to do as an after school exercise.
• Micropipet tips: Be sure that all the micropipet tip boxes are filled. These tips do not have to be kept sterile so it's OK to handle them without gloves. This task is another one that can be done by your after school students or student service person.
• Filling tubes: Fill microcentrifuge tubes (1.5 µl) with 1ml of food coloring . You can do this by using a standard medicine dropper either marked at 1ml or approximate 1ml by using 20 drops from the dropper. Use red tubes for red dye, green for green and yellow for yellow. Yellow food coloring looks very red, so color-coding the tubes keeps the students from making mistakes. Mark one microcentrifuge tube H2O and add to it 1ml of plain water. Mark one tube "Loading dye" and add 100 µl of 10X loading dye. It will take a total of 7 microcentrifuge tubes for this exercise. Three for food coloring, one for loading dye, one for water and two empty tubes. Once labeled, these can be placed in the microcentrifuge tube rack with one set for every work station.
• Preparation time: It takes about 1 hour to measure out and melt the agar. Preparation time for pouring gels should be 15-30 minutes. The time for marking and loading tubes should be 30-45 minutes. Total estimated prep time 2 hours.

Introduction

This exercise allows students to learn how to use a basic tool of molecular geneticists. They need this practice in order to be able to do the remaining laboratory exercises. Most beginning students are very clumsy at using tools especially ones that are operated with only one hand. They also should gain an appreciation for the small volume of material needed to conduct molecular genetic experiments.

Student Objectives

• The student will correctly use the micropipet.
• The student will mix liquids in a microcentrifuge tube.
• The student will load a gel.
• The student will convert measurements between the units microliters (µl) and milliliters (ml).

Class Time Needed

One 50-55 minute class period is required to do this exercise.

Materials

1. Digital micropipet
2. Tubes of H₂O and loading dye (10X)
3. Box of micropipet tips
4. 2% Agar gel (12 or more wells)
5. Tubes of food coloring
6. Empty microcentrifuge tubes (1.5µl)
7. Waste container
8. Large finger bowl
9. Microcentrifuge tube rack
10. 1/2 inch masking tape

Recipes for Consumables

2% agar is made by melting 2 g powdered agar melted in 100 ml of water. Since these gels are only being used to learn the micropipet, they can be made with tap water and not distilled water.

All other consumables are used as they come from the stock bottle.

Procedure

For the measurements portion, have the students read and then fill in the blanks. You can have students come to the front and explain how they got their answers or you can go about the room checking individual answers. Some time should be spent here but there is no need to over-emphasize this step.

Have the students read the "Don'ts", "Dialing", "Drawing Up" and "Dispensing" sections first. Then model all these steps before the students are allowed to actually begin the exercise at the "Withdrawing and Dispensing" step.

When practicing the "Loading a Gel" portion, seeing the wells in the gel is very difficult . The best thing is to place black paper under the finger bowl. To prevent the paper from getting wet, place it in a plastic liner or cover it with plastic wrap. If your lab counter tops are black in color, you can ignore this suggestion. When students are loading the gel, the most common mistake will be placing the micropipet tip too far into the well. The tip will actually be pushed into the gel, plugging it with agar and making it very difficult to expel the food coloring. You need to warn the students about this mistake many times. With practice, they will learn how deep to place the micropipet tip. Be sure that students load all red lanes before changing tips, then all green, then all yellow. This is done to save on tips. If you have an ample supply of tips, you can tell the students to eject the tip each time before drawing up and loading a new sample.

The "Mixing Liquids" section is a vital technique to learn for all future labs using the micropipet. When placed into a liquid, DNA and other dense molecules have the tendency to settle to the bottom of the container. Using this technique of mixing liquids before the student removes a sample from a microcentrifuge tube will ensure that the student actually transfers the desired material in the correct concentration.

Disposal

• Agar can be placed directly in the garbage. It is probably best to put all the gels in a zip lock bag before placing them in the garbage.
• Micropipet tips can be washed in a soap solution, rinsed and dried for reuse with this lab.
• Tubes containing loading dye and food color can be saved for future use.