Restriction Enzyme Analysis of DNA- Teacher Guide

Classroom time needed for this lab:

• three class periods (about fifty minutes each)

You will need the following equipment and supplies:

Setting up the classroom for this lab:

Distribute at each lab station (four students per group):

Day 1:

• Micropipets and tips
• Hind III restriction enzyme (6 ul in small microcentrifuge tube)
• Eco RI restriction enzyme (6 ul in small microcentrifuge tube)
• Deionized or distilled water (15 ul in small microcentrifuge tube)
• Lambda DNA (30 ul in small microcentrifuge tube)
• 4 empty microcentrifuge tubes (one restriction enzyme reaction per student)

Day 2:

• Micropipets and tips
• Electrophoresis unit and power supply (Label each electrophoresis unit with a number written on a piece of tape.)
• 6X loading dye (30 ul in small microcentrifuge tube)

Day 3:

• Small container, such as a small Rubbermaid container, to use as a methylene blue staining tray
• Methylene blue solution
• Light boxes

We use a central station for agarose and 1X TAE. We use a hot water bath to hold four small bottles, each containing 125 ml of 0.8% agarose. The high temperature keeps the agarose liquified. At room temperature we have four large bottles, each containing 1000 ml of 1X TAE.

During the lab:

Day 1:

If necessary, demonstrate to students how to use the micropipet. They can practice with deionized or distilled water before handling the reagents for the restriction enzyme reaction.

Day 2:

Be sure the students record the number of their electrophoresis unit on their lab handout. This will help students find their agarose gel during the next class.

Day 3:

When students stain their gels with methylene blue, they need to work carefully. The methylene blue can stain their clothes and skin. During destaining, the water rinse can be disposed in the sink.

After the lab:

Day1:

Students will have time to set up the restriction enzyme reactions, but not to stop the reactions. After 1.5 - 2 hours have passed, remove the reaction tubes and pool the liquid at the bottom of the tubes. Store the reaction tubes in the refrigerator until the next class.

Day 2:

After the electrophoresis units complete their run, turn off the power supply and store the gels in the refrigerator when time permits. The gels can sit in the 1X TAE for awhile. After the gels are wrapped in saran wrap and stored in the refrigerator (be sure to put masking tape on the saran wrap and record the electrophoresis unit number), the 1X TAE in the electrophoresis units can be poured back into the large bottles for reuse. Rinse the electrophoresis units with water. The units should have a final rinse with deionized or distilled water before being put away for a long period of time.

Day 3:

The used gels can be disposed or taken home by the students. If the students want the gels, cover them with saran wrap.

The methylene blue solution can be reused up to five times.

Other notes:

1. Be sure to calculate how much agarose you will need for the lab. You can add a defined volume of water into the gel tray to determine the volume of agarose that would be needed for each tray.
   
   
2. Be sure to calculate how much 1X TAE you will need for the lab. Using a graduated cylinder, you can pour a defined volume of water into the electrophoresis unit to determine the volume of 1X TAE that would be needed for each unit.
   
   
3. Recognition and cleavage sites used by some common restriction enzymes:
   
   

   Bam HI	G|G-A-T-C-C
   	C-C-T-A-G|G
   Eco RI	G|A-A-T-T-C
   	C-T-T-A-A|G
   Hind III	A|A-G-C-T-T
   	T-T-C-G-A|A
   Pst I	C-T-G-C-A|G
   	G|A-C-G-T-C

Recognition sequences are written 5' to 3' on the top strand. The " | " indicates location of cleavage site.

Recipes

1X TAE

TAE is the buffer used in gel electrophoresis.

Procedure
1. Put 48.4 g Tris base, 11.4 ml glacial acetic acid, and 20 ml of 0.5 M EDTA into a 1000 ml volumetric flask or graduated cylinder.
   
   
2. Add distilled water to make a total volume of 1 liter. This forms a 10X stock solution.
   
   
3. Dilute the 10X stock solution to make a 1X working solution (100 ml of 10X stock to 900 ml of distilled water).
   
   
4. Store 1X TAE buffer solution in a large container. The 1X TAE can be kept indefinitely at room temperature. This buffer can be reused several times before disposing down the sink.
   
   

0.8% Agarose

Agarose is the gel matrix used separating molecules, such as DNA or dyes, during electrophoresis.

1. Weigh 1 gram of agarose on a folded piece of weighing paper and add to empty 250 ml bottle or flask.
   
   
2. Add 125 ml of 1X TAE buffer solution to agarose. Note: The container should never be filled more than half-way in order to prevent the solution from boiling over.
   
   
3. If a bottle is used, cap the bottle loosely to release air during boiling. If you using a flask, cover the opening and neck of flask with plastic wrap.
   
   
4. Mix solution by swirling. Microwave the agarose solution at high heat until powder is completely dissolved. The length of time required will vary depending on the microwave oven. The molten agarose solution should look clear.
   
   
5. To keep the agarose liquified (for example, during several biology classes), store the bottle or flask of agarose in a hot water bath between 600 and 700C. Be sure that the bottle or flask is covered to prevent evaporation. A hot plate with a pot of water can substitute for a laboratory water bath.
   
   
6. If there is agarose left over in the container, you can let the agarose solidify and store it at room temperature until next use. Be sure that the container is well covered.
   
   

6X Loading Dye

This loading dye is added to the DNA sample after restriction enzyme digestion. Loading dye is important because 1) it contains sucrose or glycerol which increases the density of the DNA sample so that it sinks to the bottom of the well, and 2) it contains dyes that can indicate how far the DNA samples have run during electrophoresis.

Prepare 2% bromophenol blue stock solution:

1. Measure 0.2 g bromophenol blue powder on a square piece of folded weighing paper.
   
   
2. Using the crease in the paper, pour the dye powder into a tube. The tube should be able to hold between 10 and 15 ml.
   
   
3. Add 10 ml deionized or distilled water to the dye and cap the tube. Shake the tube well to dissolve the dye in the water. The powder may not completely dissolve in water - be sure to shake the solution well before using.
   
   
4. The 2% dye stock be stored indefinitely at room temperature.
   
   

Prepare 2% xylene cyanol stock solution:

1. Measure 0.2 g xylene cyanol powder on a square piece of folded weighing paper.
   
   
2. Using the crease in the paper, pour the dye powder into a tube. The tube should be able to hold between 10 and 15 ml.
   
   
3. Add 10 ml deionized or distilled water to the dye and cap the tube. Shake the tube well to dissolve the dye in the water. The powder may not completely dissolve in water - be sure to shake the solution well before using.
   
   
4. The 2% dye stock be stored indefinitely at room temperature.
   
   

Prepare 50% glycerol solution

1. Pour 10 ml glycerol into a 25 ml graduated cylinder (note: avoid using a pipet since glycerol is very viscous). Next, add 10 ml deionized or distilled water to the same cylinder. Use a stirring rod (a pipet would also work) to mix the glycerol and water. The glycerol solution can be stored in a small, closed container in the refrigerator.

   Prepare 6X Loading dye

   1. In a small tube with a cap, mix the following ingredients:

      6.0 ml	50% glycerol
      1.0 ml	2% bromophenol blue solution
      1.0 ml	2% xylene cyanole solution
      2.0 ml	deionized or distilled water

   2. The 6X loading dye solution can be stored indefinitely in the refrigerator.
      
      

   Practice Samples

   1. In a small tube with a cap, mix the following ingredients:

      2.0 ml	6X loading dye concentrate
      10.0 ml	deionized or distilled water

   2. The practice sample dye solution can be stored indefinitely in the refrigerator.
      
      
   3. Individual samples are prepared from this large batch by using a micropipet to measure 12 ul into a small microcentrifuge tube (0.5 or 0.65 ml size).
      
      

   0.025% Methylene blue staining solution

   Equipment and supplies	Chemicals
   Balance and weighing paper	Methylene blue trihydrate powder
   500 or 1000 ml graduated cylinder	Deionized or distilled water
   1000 ml graduated cylinder
   Magnet stirrer and stir bar
   Containers for storing methylene blue

   1. Weigh 1 g of methylene blue trihydrate powder (m.w. 373.9) in 100 ml deionized or distilled water. Stir until powder is dissolved in water. This is a 1% methylene blue concentrate, which can be stored indefinitely at room temperature.
      
      
   2. Add 10 ml 1% methylene blue concentrate to 390 ml deionized or distilled water. Stir until methylene blue is evenly mixed in solution. This solution is 0.025% methylene blue, which can be stored indefinitely at room temperature.
      
      
   3. The methylene blue solution can be reused four or five times.
      
      
   
   
   

   Sources of laboratory materials

   Item	Supplier and catalog no.
   Agarose	Life Technologies 15510-019
   Graduate micropipet tips, 250/pack	Life Technologies 10245-082
   Graduate micropipets, 5/pack	Life Technologies 10245-066
   Horizon 58 horizontal gel electrophoresis apparatus	Life Technologies 41060-013
   Life Technologies Electrophoresis Power Supply	Carolina P7-21-3700
   Microfuge tubes, 0.5 ml, 1000 per box	VWR 20170-310
   Microfuge tubes, 1.7 ml, 500/box	VWR 20170-331
   *Precision utility water bath	VWR 13470-030
   Corning hot plate/stirrer	VWR 33920-219
   White light transilluminator and AC adaptors	Carolina K3-21-6214, K3-216216
   Tris base, 1kg	VWR JT4099-2
   Boric acid. 500 g	VWR JT4035-1
   EDTA, disodium salt, dihydrate, 100 g	Life Technologies 15576-010
   Ohaus balance, CT series 200g	VWR 11378-059
   Methylene blue trihydrate powder, 25 g	Sigma M9140
   Bacteriophage Lambda DNA, 500 ug	Life Technologies 25250-010
   EcoRI restriction enzyme, 5000 units	Life Technologies 15202-013
   Hind III restriction enzyme, 5000 units	Life Technologies 15207-012
   Glycerol, 500 ml	Sigma G7893
   Bromophenol blue	Sigma B5525
   Xylene Cyanole	Sigma X4126

   *Hot plates can be substituted

   
   
   
   

   DATASHEET

   Link to the Worksheet/Datasheet for the Restriction Enzyme Analysis of Lambda DNA lab

   
   
   
   

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   BIOTECH Project Department of Molecular and Cellular Biology The University of Arizona August 3, 1998 Designed by: Ken Kubo, Ph.D. Nadja Anderson, Ph.D. nadja@email.arizona.edu http://biotech.biology.arizona.edu