Ellen Averill
1993 Woodrow Wilson Biology Institute


  1. Escherichia coli K-12 strains MM294, HB101, or the JM series are suggested as host bacteria. Suggested vectors for the DNA are pAMP. pKAN, pUC, pBR322 and M13. Suggested DNA inserts are bacteriophage lambda, T4, and E. coli sequences.

  2. Sterilize bottles, tools and solutions with an autoclave at 15 psi for 15 minutes at 121 degreesC. Do not sterilize DNA or bacterial cultures by autoclaving or heating.

  3. Use a 10% bleach solution to wipe down laboratory bench work areas before and after using microbes or DNA. Prepare the bleach solution fresh each day. Decontaminate work surfaces at least once per day and after any spills.

  4. Dispose of bacteria by placing in a biohazard bag and autoclaving at 15 psi for 30 minutes at 121deg.C or by soaking in a 10% bleach solution for one hour.. Decontaminate all contaminated liquid or solid wastes before disposal by autoclaving at 15 psi at 121deg.C. for 30 minutes or by soaking in 10% bleach solution for one hour. When autoclaving materials for disposal, loosen bottle caps and open bags so steam can escape.

  5. DNA, microorganisms and chemicals should not be stored in refrigerators with food or beverages.

  6. DNA should be kept frozen in a non-frost-free freezer. DNA should not be allowed to defrost between uses, as this will break long molecules.

  7. Bacteria can be stored as slants or stabs at room temperature; store sealed plates in a refrigerator. New cultures of bacteria should be propagated at least monthly to have live cells.

  8. If bacteria or DNA are spilled, wear gloves and absorb the spill with paper towels or bench protector paper. Working from the outside of the spill, apply disinfectant or 10% bleach solution and wipe toward the center of the spill.


  9. It is recommended that DNA isolation procedures be chosen that avoid the use of chloroform or phenol. Chloroform is toxic and phenol burns the skin on contact.

  10. Because of the possible carcinogenic and mutagenic effects of ethidium bromide, National Association of Biology Teachers recommends that pre-college level teachers use methylene blue to stain gels. Students should not use either ethidium bromide or handle gels stained with ethidium bromide. Since methylene blue is mildly toxic, gloves should be worn at all times when staining gels.

  11. Methylene blue can be used up to four times before disposal. Check with local waste management authorities before disposing of the used methylene blue.

  12. Perform all procedures to minimize the creation of aerosols. This includes not forcing the last drop of liquid from the end of a pipette..

  13. Avoid skin contact with ethanol and isopropyl alcohol apparatus.


  14. Use care when working near an open flame. Keep flammable solutions away from flames. Cover alcohol containers when not in use. Shake excess alcohol off spreaders before flaming. Hats and dangling earrings should not be worn in the lab and long hair should be pulled back.

  15. If you have cuts on your hands, wear latex gloves for protection.

  16. Each student should have and wear his/her own safety goggles. Goggles should have the logo z87 which indicates that the goggles have been approved by the American National Standards Institute and OSHA. If goggles for individual students are not available, individually wrapped alcohol pads should be used to clean goggles between uses.

  17. Wear a lab coat or apron to prevent contamination or soiling of clothes.

  18. Wear goggles approved for use with ultraviolet light when working with UV sources.

  19. Use a disposable dust mask and gloves when weighing out components of culture media and when weighing and preparing antibiotic solutions. When preparing solution of sodium dodecyl sulfate (SDS) for isolating bacteria, wear a dust mask since SDS is an irritant.


  20. Keep classroom doors closed during laboratory periods.

  21. Used razor blades should be discarded in a designated container. A metal can with a slit in the lid makes a good container for used blades.

  22. Whether using purchased or home-made gel boxes, avoid contact with the buffer solution when the chamber is running. Most electrophoresis chambers use 50 to 100 volts and are capable of delivering a shock if contact is made with the buffer while the apparatus is in operation. Safety features that prevent contact with the buffer while in operation should be part of the design of the apparatus.


  23. No food or drink should be allowed in the laboratory. Students should not chew gum or apply cosmetics and should keep all objects out of their mouths when working with microbes.

  24. Wash hands before and after doing laboratory work.

  25. Do not mouth pipette anything.

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