Team:NCTU Formosa/Safety

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The most widely used gel system for separating a broad range of proteins by SDS-PAGE is the Laemmli system (1970) which uses tris-glycine gels comprised of a stacking gel component (which is used to help focus the proteins into sharp bands at the beginning of the electrophoretic run) and the resolving gel where varying acrylamide gel percentages are used to separate the proteins based on their mass weight.  This classic system uses a discontinuous buffer system where the pH and ionic strength of the buffer used for running the gel (Tris pH 8.3) is different from the buffers used in the stacking gel (Tris, pH 6.8) and resolving gel (Tris, pH 8.8). <sup>(9)</sup>
The most widely used gel system for separating a broad range of proteins by SDS-PAGE is the Laemmli system (1970) which uses tris-glycine gels comprised of a stacking gel component (which is used to help focus the proteins into sharp bands at the beginning of the electrophoretic run) and the resolving gel where varying acrylamide gel percentages are used to separate the proteins based on their mass weight.  This classic system uses a discontinuous buffer system where the pH and ionic strength of the buffer used for running the gel (Tris pH 8.3) is different from the buffers used in the stacking gel (Tris, pH 6.8) and resolving gel (Tris, pH 8.8). <sup>(9)</sup>
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Revision as of 17:30, 17 October 2014

Safety

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Contents

Researcher Safety

1. Everyone must wear lab coat, trousers, gloves, surgery masks and shoes when carrying out experiments.

2. Before the commencement of our project, our team instructors and advisers have guided us to go through every experiment to make sure everybody is well trained and fit for this project.

3. Everybody has to sterilize his/her hands and all the equipment platforms with alcohol solution(70%) before and after each experiment.

4. Emergency equipment is well prepared and all the researchers understand how to use these equipment and their exact position.

5. It is not allowed that researcher carries experiment alone. There must be at least two members to conduct the experiment.

6. Food and beverages are not allowed to appear in the lab.


Public Safety

1. All collected lab waste are sterilized, packaged and executed.

2. E.coli used in our lab is less competitive than wild type, because they are unable to form biofilms and to thrive in the intestine.

3. Liquid waste that contains E.coli is disinfected by adding peroxides and cleaners before it is disposed.

Lab Instruments

1. Centrifuge

Fig.8-1-1 Centrifuge

A centrifuge is a kind of instrument generally driven by an electric motor. By rotating in very high speed, it produces a big centrifugal force perpendicular to the axis and rotates an object around a fixed axis. A centrifuge is used to separate the components which have different molecular weight in a mixture. The centrifuge works with the sedimentation principle, where the centripetal acceleration causes denser substances to separate out along the radial direction (the bottom of the tube). Conversely lighter objects will tend to move to the top(1). In our lab, we usually separate E.coli from LB broth and then conduct mini-prep with it.

2. Incubator

Fig.8-1-2 Incubator

The orbital shaker incubator is widely used in microbiology labs, cell biology labs, etc. It provides a stationary temperature to cultivate bacteria and cells. Some incubator also have functions to maintain the humility level. In our lab, the incubator provides stationary temperature at 37 degrees Celsius, which is the optical temperature of E.coli growing, and 16 degrees Celsius for ligase working(2). In this year, our team was authorized to work with an non-pathogenic bacterial strain, the Escherichia coli DH5. The strain is regularly employed in research, industry and study. Biosafety Level 1 standard regulations were strictly followed while using these strains.

3. Autoclave

Fig.8-1-3 Autoclave

An autoclave is a pressure chamber used to sterilize equipment and supplies by subjecting them to high pressure saturated steam at 121 °C for around 15–20 minutes depending on the size of the load and the contents. The autoclave is used for the safety issue. When sterilizing, the pressure and temperature inside reached high level, forming superheat steam and high pressure which will kill cell and make proteins destroyed or denatured(4).

In our lab, every consumables that may contact bacteria will be sterilized before and after use to guarantee the safety.

4. Laminar Flow

Fig.8-1-4 Laminar flow

A laminar flow is an enclosed, ventilated laboratory workspace for safely working with materials contaminated with (or potentially contaminated with) pathogens requiring a defined biosafety level. We usually conduct experiments where our E.coli will expose, such as selecting single colony(7). We strictly rule that all of the researchers should conduct experiments carefully inside the laminar flow and sterilize his/her hands with alcohol solution(70%) before conducting experiments, all of us are familiar with the using method of Laminar flow.

5. Balance

Fig.8-1-5 Balance

An analytical balance is a class of balance used in measuring small mass in the sub-milligram range. The measuring pan of an analytical balance is inside a transparent enclosure with doors so that dust can be collected well and so any air currents in the room do not affect the balance's accuracy(5). In our lab, we have two models of balance, one is accurate to 0.1 g, another is accurate to 0.1 mg.

6. Water Purify System

Fig.8-1-6 Water purify system

In this system, water is purified in a first step using unique Jetpore ion-exchange resin, synthetic activated carbon and UV lamp emitting at 185 and 254 nm to reach a resistivity of 18.2 MΩ.cm at 25 °C and a TOC value below 5 ppb, both monitored by advanced analytical techniques(6). The water purify system is used to produce ultrapure water, which is for experimental use in our lab.

7. Thermal Cycler

Fig.8-1-7 Thermal cycler

The thermal cycler (also known as a thermocycler, PCR machine or DNA amplifier) is a laboratory apparatus that are most commonly used to amplify segments of DNA via the polymerase chain reaction (PCR). The function of thermal cycle is to provide the appropriate working temperature for some reactions, including many temperature-sensitive reactions such as restriction enzyme digestion or rapid diagnostics. The device has a thermal block with holes where tubes holding the reaction mixtures can be inserted. The cycler then raises and lowers the temperature of the block in discrete, pre-programmed steps, and time setting determines the time interval of every temperature(3).

8. Gel Documentation System

Fig.8-1-8 Gel documentation system

Gel documentation system, also known as a gel image system or gel imager, is equipment widely used in molecular biology laboratories for the imaging and documentation of nucleic acid and protein suspended within polyacrylamide or agarose gels. These gels are typically stained with ethidium bromide or other fluorophores such as SYBR Green. Generally, a gel doc includes an ultraviolet (UV) light transilluminator, a hood or a darkroom to shield external light sources and protect the user from UV exposure, and a CCD camera for image capturing(8).

9.Protein Gel Electrophoresis Equipment

[[File:Protein Gel Electrophoresis Equipment .jpg|200px|thumb|center|Fig.8-1-9 Protein Gel Electrophoresis Equipment]

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) is a technique for separating proteins based on their ability to move within an electrical current, which is a function of the length of their polypeptide chains or of their molecular weight. This is achieved by adding SDS detergent to remove secondary and tertiary protein structures and to maintain the proteins as polypeptide chains. The SDS coats the proteins, mostly proportional to their molecular weight, and confers the same negative electrical charge across all proteins in the sample. Glycosylated proteins may not migrate at their expected molecular weight since their migration is based more on the mass of their polypeptide chains, not the sugars that are attached (Sambrook, etal, 1989).

The most widely used gel system for separating a broad range of proteins by SDS-PAGE is the Laemmli system (1970) which uses tris-glycine gels comprised of a stacking gel component (which is used to help focus the proteins into sharp bands at the beginning of the electrophoretic run) and the resolving gel where varying acrylamide gel percentages are used to separate the proteins based on their mass weight. This classic system uses a discontinuous buffer system where the pH and ionic strength of the buffer used for running the gel (Tris pH 8.3) is different from the buffers used in the stacking gel (Tris, pH 6.8) and resolving gel (Tris, pH 8.8). (9)


Reference
  1. Biological Centrifugation, by D. Rickwood, J.M. Graham (2001). Springer Verlag; ISBN: 0387915761
  2. Incubator (culture), from Wikipedia, http://en.wikipedia.org/wiki/Incubator_(culture)
  3. Weier, HU; Gray, JW (Jul–Aug 1988). "A programmable system to perform the polymerase chain reaction. " DNA (Mary Ann Liebert, Inc. ) 7 (6): 441–7. PMID 3203600
  4. Seymour Stanton Block (2001). Disinfection, Sterilization, and Preservation. Lippincott Williams & Wilkins. ISBN 978-0-683-30740-5. Retrieved 19 January 2013
  5. "A&D training material" by Marketing Department A&D Company. Analytical balance, from Wikipedia , http://en.wikipedia.org/wiki/Analytical_balance
  6. Milli-Q Direct, made by EMD Millipore, http://www.millipore.com/catalogue/module/C85358
  7. "The Safe Use of Biological Safety Cabinets" by Environmental Health and Radiation Safety
  8. E-BOX VX5 Gel Documentation System, made by Vilber Lourmat, http://www.fisherbiotec.com.au/shop/product/e-box_vx5_gel_documentation_system/3. Gel Documentation System, from Wikipedia, http://en.wikipedia.org/wiki/Gel_doc
  9. Suvra Roy, Vikash Kumar, A Practical Approach on SDS PAGE for Separation of Protein, International Journal of Science and Research (IJSR).
  10. Sambrook, J, Fritsch, EF, Maniatis, T (1989) Molecular Cloning: A Laboratory Manual, 2nd Edition. Cold Spring Harbor Press NY pp18.47-18.59.

Safety Form