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Appendix B: Commonly used techniques
Sterilizing in molecular biology.
Most solutions we work with in molecular biology are sterile. The need for sterile growth
media is somewhat obvious; after all, we are trying to grow very specific organisms, not
just whatever falls out of the air. Many other solutions we use in molecular biology can
also be nice substrates for microbial growth. When we isolation plasmid, or run a gel, we
prefer to look at DNA from the bacteria we were using as a cloning vehicle, not the DNA
of bacteria or fungi that are just lying around the lab. Even small amounts of
contamination can cause big problems, especially if you will be using amplification
techniques, such as PCR, downstream.
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.
Additionally, before disposal we are required to decontaminate the following: all
genetically modified organisms, anything contaminated with such organisms, or nucleic
acids from such organisms. In other words, pretty much anything we work with should be
treated as a biohazardous waste. The easiest and most affective way to decontaminate
most waste is by autoclaving. Exceptions include human and animal tissue, which
should be incinerated for disposal, and items contaminated by volatile organics, such as
phenol, or chloroform, which should go to a certified chemical disposal plant.
Sterilizing by autoclaving.
Autoclaving, sometimes called steam sterilization, is the use of pressurized steam to kill
infectious agents and denature proteins. Water is heated to temperatures above its
boiling point (to a minimum of 120 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 slightly 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 PP (polypropylene) or
polycarbonate (PC) are the best choices.
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Autoclaving liquids
1. Loosely set caps on bottles, or cover openings with foil or foam plugs.
2. 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.
3. Only after solutions have completely cooled, tighten the lids.
Decontaminating solutions containing ethidium bromide.
There are a variety of ways to remove ethidium bromide from solution, primarily gel
running buffers. All of them involve binding up the ethidium bromide with some type of
solid. Once decontaminate, 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 which will bind ethidium bromide include:
activated charcoal
certain resins
Autoclaving plastics, tips in tip boxes, glass etc.
1. Wrap plastics, glass etc. in foil, PP bags, or place in appropriate sized plastic or
pyrex containers.
2. Loosely set caps on any containers.
3. 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.
4. Only after containers have completely cooled, tighten the lids.
How to filter sterilize
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.
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