Producing a 1.0%, 2.0%, 3.0% and 4.0% Agarose Gel Solution
- 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
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
- 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)
- Laboratory balance
- 70 ml 1X TBE buffer
- 250 ml beaker
- burner, ring stand, wire gauze or hot plate
- zip lock sandwich bags
- 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.
- 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.
- 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.
- Tare a 100 ml beaker on a balance.
- Pour the correct number of grams (# of grams you calculated below)
of TBE buffer into the 100 ml beaker.
- Tare the 100 ml beaker with the TBE buffer in it on a balance.
- Pour the correct amount of agarose into the beaker, stirring constantly.
- 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.
- 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.
- When the agarose is dissolved, (the solution will turn clear, be patient)
pour 30 ml into each tray.
- 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.
Total mass of solution equals 70.0 grams. Fill in the chart below and
show your work.
||Mass of TBE Buffer
||Mass of Agarose
- 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
- When the agarose is added to the buffer, is it a suspension or solution?
How could you tell? SUSPENSION- SOLUTION IS CLOUDY
- 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
- 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