Producing a 1.0%, 2.0%, 3.0% and 4.0% Agarose Gel Solution

Advance Preparation

• Two days before the lab, have students do "Making solutions" worksheet. ¥ One day before the lab demonstrate to students the correct procedure for: (1) dissolving agarose in water, (2) tarring a beaker, (3) taping a gel tray, (4) setting a gel comb, (5) pouring hot agarose into a gel tray, (6) removing a comb and storing gels in plastic bags.
• The agarose must be made using 1XTBE buffer NOT water.
• Gels: The volume of 1X TBE buffer needed for each pair of students to make 2 gels is approximately 80 mls. You will need this volume times the number of pairs of students doing this exercise. 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 more leaks than trying to fold the tape under and attaching it to the bottom of the tray. Pouring the agarose should be done at approximately 60-65 oC. This can be approximated by feel. If you can hold both hands around the beaker and not get a burning sensation, the agarose is at the correct temperature. You should have as many gel trays prepared as you have agarose . Each gel requires 30-35 mls of agarose. The wells need to be in the center of the gel so be sure to set the gel comb in the center of the tray. They can be stored inside zip lock bags at room temperature. Each student will pour one extra gel, If the students make major mistakes they can use their back-up to complete the exercise. Since this lab is designed to get results within 20 minutes, it is essential that the students begin pouring the gels as soon as possible.
• Placing about 20 grams of dry agarose into baby food jars will speed up the dispensing process. One jar per two teams of 2 students each should be sufficient.

Introduction

Agarose gels are often used in electrophoresis equipment as the media on which DNA is separated. The density of these gels greatly determines how fast the DNA migrates down the track. The greater the density, the slower the rate of migration. (For much the same reason that it would take you longer to swim through corn syrup than it would through water.) In a gel solution, the solute is agarose (a seaweed product) and the solvent is TBE buffer. Typically, gels are poured in a range of 1.0% to 4.0% agarose by mass. In this lab students will calculate the number of grams of agarose necessary to make 70.0 ml of EITHER a 1.0%, 2.0%, 3.0% or 4.0% agarose gel solution. They will then dissolve the solute in the solvent and pour the gel.

Student Objectives

• Students will correctly calculate the mass of solute and solvent necessary to make a 1%, 2%, 3% and 4% solution.
• Students will use proper laboratory procedure to make either a 1%, 2%, 3% or 4% solution.
• Students will, using techniques observed from the previous days demonstration, make and pour their own electrophoresis gel.

Class Time Needed

One 55 minute period

Materials (per pair of students)

1. Laboratory balance
2. 70 ml 1X TBE buffer
3. 250 ml beaker
4. burner, ring stand, wire gauze or hot plate
5. zip lock sandwich bags
6. two gel trays and combs from the electrophoresis equipment

Recipes for Consumables

1XTBE buffer is made by diluting 100 mls of 10XTBE stock solution in 900 mls of distilled water.

Procedure

1. Calculate both the mass of agarose and the mass of TBE buffer which should be mixed in order to make 70.0 grams of 1.0%, 2.0%, 3.0% or 4.0% mixtures. Show your work on the calculations portion of this sheet, and place your answers in the chart provided on the next page. CHECK THE STUDENT'S WORK AND BE SURE IT MATCHES THE CHART.
2. Have your instructor check your work, then assign you one of the mixtures. ASSIGN STUDENTS EITHER A 1.0%, 2.0%, 3.0% or 4.0% MIXTURE. TRY TO ASSIGN THE SAME NUMBER OF EACH MIXTURE.
3. Tare a 100 ml beaker on a balance.
4. Pour the correct number of grams (# of grams you calculated below) of TBE buffer into the 100 ml beaker.
5. Tare the 100 ml beaker with the TBE buffer in it on a balance.
6. Pour the correct amount of agarose into the beaker, stirring constantly.
7. Carefully heat the TBE buffer on a hot plate INSTRUCT THE STUDENTS NOT TO LET THE SOLUTION GET TOO HOT OR IT WILL BOIL OVER.
8. While one partner is stirring the solution, the other partner should prepare the gel tray as instructed by your teacher. INSTRUCT THE STUDENTS TO CONSTANTLY STIR THE SOLUTION OR IT WILL BURN AND STICK TO THE BOTTOM OF THE BEAKER.
9. When the agarose is dissolved, (the solution will turn clear, be patient) pour 30 ml into each tray.
10. At the end of the period, remove the comb from the gel and carefully slide the gel into a plastic bag, labeled with your name on it.

Analysis

Total mass of solution equals 70.0 grams. Fill in the chart below and show your work.

Conclusion

1. TBE buffer is a series of salts dissolved in water. Will TBE buffer boil above 100^oC or below 100^oC at 1 atm of pressure? What constant could be used to determine how much change would occur in the boiling point of TBE buffer? ABOVE- MOLAL BOILING POINT ELEVATION OF WATER
2. When the agarose is added to the buffer, is it a suspension or solution? How could you tell? SUSPENSION- SOLUTION IS CLOUDY
3. Is the powdered agarose likely to be a polar or non-polar molecule? What evidence can you give to support your answer? SLIGHTLY POLAR- IT DISSOLVES IN WATER, BUT NOT EASILY
4. After the powder is dissolved in hot water and allowed to cool, what happens to its polarity? What evidence can you give to support your answer. GETS LESS POLAR- WON'T DISSOLVE IN WATER AS EASILY