The following materials are based upon 12 groups per class with 2-3 students in each group. Most clinical centrifuges hold six 15 mL tubes; all the groups would be accommodated by a series of two cycles.

#	Item	#	Item
24	plastic 15 mL tubes with lids *	750 mL	Gatorade or saline solution
40	small paper cups (Dixie cups)	50 mL	lysis buffer
48	1 mL disposable pipettes *	40 mL	5M NaCl
12	1.5 mL Eppendorf tubes	75 mL	95% ethanol
6	15 mL graduated cylinder * or sterile 15 mL graduated tubes	10 mL	TE buffer (optional) or water
1 - 2	clinical centrifuge	600 uL	proteinase K (10 mg/mL)(optional)
1	hot water bath (65 - 70 C)	6-12	beakers filled with ice
1	thermometer	6-12	permanent markers
18	test tube racks	6-12	glass rod with hooks

Initially this lab takes a great deal of time to set up and prepare the chemical solutions. Many of the solutions required can be made in large volumes and stored on the shelf from year-to-year. The lysis buffer, sucrose and saline solutions need to be made fresh each time.

I provide each group with small quantities (5 - 10 mL) of the solutions they will need in labeled plastic tubes. I refill the tubes as needed, but generally 5-6 classes can use the same set of chemicals. This also keeps the students from contaminating my stock solutions.

Recipes

O.5 M EDTA pH 8.0 (disodium salt of Ethylenediaminetetraacetic Acid)

18.6 g EDTA
2.2 g NaOH pellets or concentrated NaOH solution

1. Dissolve in 80 mL distilled water.
2. Adjust pH to 8.0 by adding about 2.2 g NaOH pellets.
3. Mix vigorously with a magnetic stirrer (or by hand).
4. EDTA dissolves when the pH has reached 8.0 or higher.
5. Add distilled water to bring final volume to 100 mL.
   
   

Lysis Buffer

5 mL 1M Tris pH 8.0
10 mL 0.5M EDTA pH 8.0
5 mL 1M Sucrose
5 mL 10% SDS
2 mL 5M NaCl

1. Add distilled water to bring volume to 100 mL.
2. Mix well.
   

• Lysis buffer needs to be made up fresh each time you do this lab because bacteria will start to grow (the sucrose provides nourishment).
• Check for contamination by swirling the container and looking for floating particles or bacterial threads.
  
  
  

Proteinase K (optional)

25 mg proteinase K
2.5 mL distilled H₂O

1. Add the water to the vial containing the proteinase K powder.
2. Swirl to dissolve.
3. Freeze proteinase K solution between uses.
   
   
   

0.9% Saline Solution or use Gatorade

9 g NaCl
1 L distilled H₂O

1. Mix in a clean container because will be putting the saline solution in their mouths.
2. Swirl to dissolve.
   

• Gatorade needs to be purchased fresh each time. It will start to grow bacteria if stored open for longer than a few days to a week.
• Check for contamination by swirling the container and looking for floating particles or bacterial threads.
• I prefer to use Gatorade because I know it's sterile. Choose a light color flavor, such as lemon, to keep from staining the DNA.
  
  
  

5 M Sodium Chloride (NaCl)

29.2 g NaCl
distilled water

1. Dissolve 29.2 g of NaCl in 80 mL of water over low heat using a magnetic stirbar.
2. This is almost a saturated solution. It will take a long time for the NaCl to dissolve completely.
3. Bring final volume to 100 mL.
   

1 M Sucrose

8.55 g sucrose
distilled water

1. Dissolve sucrose in 15 mL of distilled water over low heat.
2. Bring final volume to 25 mL with distilled water.
   

• Sucrose solution needs to be made fresh each time you do this lab or frozen between uses to prevent bacterial contamination.
• To check for contamination, swirl the bottles containing sucrose or lysis buffer and look for floating particles and bacterial strands.
  
  
  

10% SDS (Sodium Lauryl Sulfate)

10 g electrophoresis grade SDS
distilled water

1. Add 80 mL of distilled water.
2. Dissolve SDS completely.
3. Add distilled water to bring final volume to 100 mL.
   

• Avoid inhaling SDS powder; wear a mask over mouth & nose.
• SDS solution can be stored over long periods of time at room temperature.
  
  
  

1 M Tris (pH 8.0)

12.1 g Tris base
concentrated HCl
distilled water

1. Dissolve in 70 mL distilled water.
2. Adjust pH to 8.0 by adding about 5.0 mL of concentrated HCl.
3. Add distilled water to bring final volume to 100 mL.
   

• pH is temperature dependent; measure the pH at room temperature.
• Avoid inhaling Tris powder; wear a mask over your mouth and nose.
  
  

Tris-EDTA (TE) Buffer

1 mL 1M Tris (10 mM in solution) concentrated HCl
200 uL 0.5 M EDTA (1 mM in solution) concentrated HCl
99 mL distilled water

• TE buffer is used by research laboratories to store and protect DNA over long periods of time.
• For our purposes, DNA can also be dissolved in water and studied by agarose gel electrophoresis.

Science Education Connection Department of Biochemistry The University of Arizona Tuesday, January 14, 1997 warder@u.arizona.edu http://biology.arizona.edu/sciconn/lessons/vuturo/ All contents copyright © 1997. All rights reserved.