The success of the laboratory depends on the use of high-quality reagents, such as are available from Carolina Biological Supply Company. The recipes are organized in five parts: DNA Isolation, Polymerase Chain Reaction, Agarose Gel Electrophoresis, and Cycle Sequencing. Reagents and stock solutions are listed alphabetically within each section. Follow the recipes with care and pay attention to cleanliness. Use a clean spatula for each ingredient or carefully pour each ingredient from its bottle.

If you are just beginning or have minimum time for preparation, consider using a ready-to-use kit that contains all reagents you will need for 25 student samples. The following kit was developed in cooperation with Carolina Biological Supply Company:

Human Mitochondrial DNA Kit AT

Catalog # 21-1236 DNA Extraction and Amplification Kit includes Chelex, proteinase K, Ready-to-Go Beads (in 0.5 ml tubes), primer/loading dye mix, and DNA markers.

Catalog # 21-1237 or 21-1238 DNA Extraction, Amplification, and Electrophoresis Kit includes all of the above, plus agarose, electrophoresis buffer, and either ethidium (1237) bromide stain or CarolinaBLU stain (1238).

I. DNA Isolation

10% Chelex

Makes 10 ml. Store at room temperature (3 months).

  1. Weigh out 1 g of Chelex 100 (100-200 mesh, sodium form from BioRad).
  2. Add 50 mM Tris to dry Chelex to make 10 ml of solution.
  3. Adjust pH to 11 using 4 N NaOH.

Saline Solution, 0.9% Sodium Chloride (NaCl)

Makes 1000 ml. Store at room temperature (indefinitely).

Note: This solution will be used for a mouthwash, so insure that all glassware/plasticware is clean and free of chemical residue.

  1. Dissolve 9 g NaCl (mw 58.44) in 700 ml deionized or distilled water in clean container.
  2. Add water to bring total solution volume to 1000 ml.
  3. Make 10 ml aliquots in sterile 15 ml culture tubes.

Note: If solution is to be used immediately, it need not be sterile. However, for long-term storage, we recommend sterilizing by autoclaving for 15 minutes at 121C or passage through a .45 or .22 micron sterile filter.

M I T O C H O N D R I A L  C O N T R O L  R E G I O N

DNA Learning Center, Cold Spring Harbor Laboratory
Noncommercial, educational use only.
Kits available from Carolina Biological Supply Company.