Team:NCTU Formosa/Safety

From 2014.igem.org

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(8. Gel Documentation System)
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2. Before the commencement of our project, our team instructors and advisers have guided us to go through every experiment to make
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2. Before the commencement of our project, our team instructors and advisers have guided us to go through every <div style="margin-left:50px;">experiment to make sure everybody is well trained and fit for this project. </div></p>       
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sure everybody is well trained and fit for this project. </p>       
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3. Everybody has to sterilize his/her hands and all the equipment platforms with alcohol solution(70%) before and after each experiment.</p>
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3. Everybody has to sterilize his/her hands and all the equipment platforms with alcohol solution (70%) before and after each <div style="margin-left:50px;">experiment.</div></p>
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4. Emergency equipment is well prepared and all the researchers understand how to use these equipment and their exact position.</p>
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4. Emergency equipment is well prepared and all the researchers understand how to use these equipment and their exact <div style="margin-left:50px;">position.</div></p>
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5. It is not allowed that researcher carries experiment alone. There must be at least two members to conduct the experiment.</p>
5. It is not allowed that researcher carries experiment alone. There must be at least two members to conduct the experiment.</p>
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====1. Centrifuge====
====1. Centrifuge====
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[[File:NCTU FORMOSA 2014 WIKI SAFETY centrifuge.JPG|200px|thumb|center||Fig.8-1-1 Centrifuge ]]
 
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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<sup>(1)</sup>.
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<sup>(1)</sup>.
In our lab, we usually separate ''E.coli'' from LB broth and then conduct mini-prep with it.
In our lab, we usually separate ''E.coli'' from LB broth and then conduct mini-prep with it.
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[[File:NCTU FORMOSA 2014 WIKI SAFETY centrifuge.JPG|200px|thumb|center||Fig.1-3-1 Centrifuge ]]
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====2. Incubator====
====2. Incubator====
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[[File:NCTU FORMOSA 2014 WIKI SAFETY incubator.JPG|thumb|200px|center|Fig.8-1-2 Incubator ]]
 
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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.
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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 incubators also have function to maintain the humility level.
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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<sup>(2)</sup>.
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In our lab, the incubator provides stationary temperature at 37 °C, which is the optical temperature for ''E.coli'' to grow, and 16 °C for ligase enzyme working<sup>(2)</sup>.
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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.
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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 this strain.
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[[File:NCTU FORMOSA 2014 WIKI SAFETY incubator.JPG|thumb|200px|center|Fig.1-3-2 Incubator ]]
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[[File:2014igem NCTU FORMOSA WIKI Autoclave.JPG|thumb|200px|center|Fig.8-1-3 Autoclave]]
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[[File:2014igem NCTU FORMOSA WIKI Autoclave.JPG|thumb|200px|center|Fig.1-3-3 Autoclave]]
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[[File:NCTU Formosa 2014 Laminar flow.jpg|200px|thumb|center|Fig.8-1-4 Laminar flow ]]
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[[File:NCTU Formosa 2014 Laminar flow.jpg|200px|thumb|center|Fig.1-3-4 Laminar flow ]]
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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<sup>(7)</sup>.
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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 in which ''E.coli'' will expose, such as picking single colony<sup>(7)</sup>.
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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.
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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 experiments, all of us are familiar with the using method of Laminar flow.
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[[File:NCTU Formosa 2014 Balance.jpg|200px|thumb|center|Fig.8-1-5 Balance]]
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[[File:NCTU Formosa 2014 Balance.jpg|200px|thumb|center|Fig.1-3-5 Balance]]
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[[File:NCTU Formosa 2014 Water purify system.jpg|200px|thumb|center|Fig.8-1-6 Water purify system]]
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[[File:NCTU Formosa 2014 Water purify system.jpg|200px|thumb|center|Fig.1-3-6 Water purify system]]
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  [[File:NCTU_Formosa_2014_thermal cycle.jpg|200px|thumb|center|Fig.8-1-7 Thermal cycler ]]
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  [[File:NCTU_Formosa_2014_thermal cycle.jpg|200px|thumb|center|Fig.1-3-7 Thermal cycler ]]
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[[File:NCTU Formosa 2014 gel imager.jpg|200px|thumb|center|Fig.8-1-8 Gel documentation system]]
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[[File:NCTU Formosa 2014 gel imager.jpg|200px|thumb|center|Fig.1-3-8 Gel documentation system]]
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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<sup>(8)</sup>.
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<sup>(8)</sup>.
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====9.Protein Gel Electrophoresis Equipment====
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====           9. Protein Gel Electrophoresis Equipment         ====
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[[File:Protein Gel Electrophoresis Equipment .jpg|200px|thumb|center|Fig.8-1-9 Protein Gel Electrophoresis Equipment]]
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[[File:Protein Gel Electrophoresis Equipment .jpg|200px|thumb|center|Fig.1-3-9 Protein Gel Electrophoresis Equipment]]
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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). 
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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (called SDS-PAGE) is a technique for separating proteins based on ability to move within an electrical current, which is a function of the length of their polypeptide chains or of their molecular weight. By adding SDS detergent to remove secondary and tertiary protein structures, the proteins maintain 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.
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To help focus the proteins into sharp bands at the beginning of the electrophoretic run, the Laemmli system is widely used, which uses tris-glycine gels comprised of a stacking gel component and the resolving gel where varying acrylamide gel percentages are used to separate the proteins based on their mass weight<sup>(9)</sup>.
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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>
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<p>'''We conducted the polyacrylamide gel electrophoresis to check whether ''E.coli'' can express our PBAN device correctly.'''</p>
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<ul class="download">  
<ul class="download">  
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<li>Safety Form<p></p>[https://igem.org/Safety/Safety_Form?team_id=1415 To igem]</li>
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<li>Safety Form<p></p>[https://igem.org/Safety/Safety_Form?team_id=1415 To iGEM]</li>
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<li>Check In<p></p>[https://igem.org/Safety/Check_In?team_id=1415 To igem]</li>
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<li>Check In<p></p>[https://igem.org/Safety/Check_In?team_id=1415 To iGEM]</li>
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<li>About Our Lab<p></p>[https://igem.org/Safety/About_Our_Lab?team_id=1415 To igem]</li>
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<li>About Our Lab<p></p>[https://igem.org/Safety/About_Our_Lab?team_id=1415 To iGEM]</li>
</ul>
</ul>

Latest revision as of 03:48, 18 October 2014

Safety

Change the font size right here

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

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.

Fig.1-3-1 Centrifuge

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 incubators also have function to maintain the humility level. In our lab, the incubator provides stationary temperature at 37 °C, which is the optical temperature for E.coli to grow, and 16 °C for ligase enzyme working(2). 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 this strain.

Fig.1-3-2 Incubator

3. Autoclave

Fig.1-3-3 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.1-3-4 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 in which E.coli will expose, such as picking 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 experiments, all of us are familiar with the using method of Laminar flow.

5. Balance

Fig.1-3-5 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.1-3-6 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.1-3-7 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.1-3-8 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

Fig.1-3-9 Protein Gel Electrophoresis Equipment

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (called SDS-PAGE) is a technique for separating proteins based on ability to move within an electrical current, which is a function of the length of their polypeptide chains or of their molecular weight. By adding SDS detergent to remove secondary and tertiary protein structures, the proteins maintain 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. To help focus the proteins into sharp bands at the beginning of the electrophoretic run, the Laemmli system is widely used, which uses tris-glycine gels comprised of a stacking gel component and the resolving gel where varying acrylamide gel percentages are used to separate the proteins based on their mass weight(9).

We conducted the polyacrylamide gel electrophoresis to check whether E.coli can express our PBAN device correctly.


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