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

Advance Preparation

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

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.

Gel % Mass of TBE Buffer Mass of Agarose
1 69.3g 0.7g
2 68.6g 1.4g
3 67.9g 2.1g
4 67.2g 2.8g

Conclusion

  1. TBE buffer is a series of salts dissolved in water. Will TBE buffer boil above 100oC or below 100oC 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