Commonly used molecular techniques

Sterilizing in molecular biology

Most solutions we work with in molecular biology are sterile. The need for sterile growth media is obvious; however, most solutions used in molecular biology need to be kept sterile because they are excellent substrates for microbial growth. Even small amounts of microbial contamination can cause problems, especially if you will be using amplification techniques, such as PCR.

Keeping all solutions sterile means you can make larger batches of solutions to use over longer periods of time. Otherwise, you will find yourself preparing fresh solutions more often then you would like.

We also use only sterile consumables (e.g. tips and tubes) as biological contaminants can be carried on these.

Sterilization by autoclaving is an acceptable method for decontaminating biohazardous waste before disposal. We are required to decontaminate the following: all potentially hazardous organisms, genetically modified organisms, anything contaminated with such organisms, and nucleic acids from such organisms. In other words, pretty much everything we dispose of should be treated as biohazardous waste.

Autoclaving does not sufficiently decontaminate human or animal tissue; or volatile chemicals. Animal tissues must be incinerated. Volatile organics, such as phenol, or chloroform, must be sent to a certified chemical disposal plant.

Sterilizing by autoclaving

Autoclaving uses of pressurized steam to kill infectious agents and denature proteins. Water is heated to temperatures above its boiling point (to a minimum of 121 °C) by holding it in a pressurized chamber. This kind of "wet heat" is considered the most dependable method of sterilizing laboratory equipment and decontaminating biohazardous waste. Autoclaves do not remove chemical contamination. In fact autoclaving some chemicals can lead to production of dangerous fumes.

Packaging of items or liquids to be sterilized must permit heat (steam) penetration, and prevent pressure differentials that could result in breakage. This may be accomplished by using techniques such as:

  • loosening screw caps or using self venting caps,
  • capping open containers for sterilization with aluminum foil,
  • opening plastic bags prior to loading them into the autoclave.

Do not place sealed containers in an autoclave!
Additionally, make sure any containers you put in the autoclave can handle the heat and pressure. For glass, Pyrex is a good choice; for plastic use polypropylene (PP) or polycarbonate (PC).

Autoclaving liquids

Loosely set caps on bottles, or cover openings with foil or foam plugs.
Autoclave for 20 min at 120 °C and 15 lb/sq. in. using a slow exhaust (using fast exhaust can result in boiling over of superheated solutions). Longer times for autoclaving may be required if working with large volumes.
Only after solutions have completely cooled, tighten the lids.

Autoclaving plastics, tips in tip boxes, glass etc.

  • Wrap plastics, glass etc. in foil, PP bags, autoclavable plastic containers or Pyrex containers.
  • Loosely set caps on any containers.
  • Autoclave for 20 min at 120 °C and 15 lb/sq. in. Inert materials can be exhausted either fast or slow. Drying cycles are optional, if available they will remove most of the condensed water from the items.
  • Only after containers

have completely cooled, tighten the lids.

Filter sterilizing

Solutions that contain heat-labile components must be filter-sterilized. Such solutions include many proteins, vitamins, amino acids, carbohydrates and most antibiotics.

To filter sterilize small volumes (10 ml or less) use a filter which can be fitted on the end of a syringe. For larger volumes use sterile filters which can be fitted onto a bottle top. Porosity of the filter membrane should be no larger than 0.2 microns (µm). Collect the filtered solution in sterile containers.

Decontaminating solutions containing ethidium bromide

There are a variety of ways to remove ethidium bromide from solution. All of them involve binding the ethidium bromide with some type of solid. Once decontaminated, the remaining solution can be safely poured down the drain. The contaminated solid material is sent to a chemical waste disposal plant for further treatment.

Examples of solids that will bind ethidium bromide include:

  • activated charcoal
  • certain resins such as Amberlite XAD-16 (Sigma)

Decontaminating a liquid ethidium bromide solution with Amberlite XAD-16

(Lunn and Sansone 1987)

  1. Add 2.9 g of Amberlite XAD-16 for each 100 ml of solution up to 100 µg/ml ethidium bromide. Amberlite XAD-16, a nonionic, polymeric absorbent, is available from Rohm and Haas and sold by Sigma Chemical Company.
  2. Store the solution for 12 hours at room temperature, shaking it intermittently.
  3. Filter the solution through a Whatman No. 1 filter. Discard the filtrate to the drain.
  4. Seal the filter and Amberlite in a plastic bag and dispose of the bag through the Office of Research Safety.
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