Team:UNSW Australia/Protocols Safety

iGEM UNSW

Agarose Gel Preparation


  1. To prepare a 50mL agarose gel solution, first add 5g of agarose powder to an 100mL empty bottle before adding 50mL of 99% TAE buffer.
  2. Heat the solution in the microwave until it starts bubbling, take out and swirl until agarose powder dissolves and the solution turns clear. Repeat several times until the agarose powder has completely dissolved.
  3. Pour the agarose gel solution into a chamber with the comb set up and allow it to cool.
  4. Add 2μL of 6X dye to 10μL of DNA, mix it slightly before loading into wells.

Safety Considerations

Chemical/ Device/ Component Risk Risk Prevention
Heated Agarose gel Burns and scalds Wear protective clothing, such as heat-proof gloves, when handling the heated container.

Ampicillin Stock Preparation


  1. Weigh 57.5 g of Ampicillin trihydrate powder using an analytical balance.
  2. Add 1mL of water.
  3. Run the tube in a gravity centrifuge for 30 seconds.
  4. Vortex contents of the container until the solution turns cloudy.
  5. Gradually add NaOH until the solution becomes clear. When the solution is completely clear, stop adding NaOH. Further addition of NaOH will result in a yellow solution which will no longer be usable.
  6. Take a filter and attach a syringe to it.
  7. Add the solution to the syringe.
  8. Transfer the solution from the syringe to an Eppendorf tube.
  9. Store the ampicillin stock in the fridge (-20℃) for future use.

Safety Considerations

Chemical/ Device/ Component Risk Risk Prevention
Ampicillin trihydrate powder (ThermoFisher SCIENTIFIC, 2018). Acute toxicity, Repeated Dose Toxicity, Reproduction & Development Toxicity, Genetic Toxicity, Carcinogenicity (Carcinogen status: Group 3). Causes skin irritation, Causes serious eye irritation, May cause an allergic skin reaction, May cause allergy or asthma symptoms or breathing difficulties if inhaled. Wash face, hands and any exposed skin thoroughly after handling. Wear protective gloves/protective clothing/eye protection/face protection. Avoid breathing dust/fume/gas/mist/vapors/spray. In case of inadequate ventilation wear respiratory protection. Contaminated work clothing should not be allowed out of the workplace. Use only outdoors or in a well-ventilated area.
5M NaOH (Merck, 2017). May be corrosive to metals. Causes severe skin burns and eye damage. Prevention: Wear protective gloves/ protective clothing/ eye protection/ face protection. Response: IF SWALLOWED: Rinse mouth. Do NOT induce vomiting. IF IN EYES: Rinse cautiously with water for several minutes. Remove contact lenses, if present and easy to do. Continue rinsing. IF exposed or concerned: immediately call a POISON CENTER or doctor/ physician. When not using the solution, close the lid to avoid any spillage.
Shaking the microfuge tube. Make sure the lid is tightly closed before shaking. Wear gloves and protective goggles at all times.
Electrocution/ burns from centrifuge or vortex. Make sure that no liquids are around the devices. Make sure that the appliances are tagged before use. If unsure on how to operate them, ask the supervisors. Be careful when using the devices.

BugBuster Protein Extraction


  1. Harvest cells from liquid culture by centrifugation at 10,000 × g for 10 min using a weighed centrifuge tube. For small scale extractions (1.5 ml or less), centrifugation can be performed in a 1.5-ml tube at 14,000–16,000 × g. Decant and allow the pellet to drain, removing as much liquid as possible. Determine the wet weight of the pellet.
  2. Resuspend the cell pellet in room temperature BugBuster Master Mix by pipetting or gentle vortexing, using 5 ml reagent per gram of wet cell paste. This typically corresponds to about 2.5 ml per 50-ml. culture. For small cultures use up to 1/5 culture volume for resuspension (e.g., use 300 µl BugBuster® Master Mix for 1.5 ml cultures). There are no adverse effects to using larger volumes of BugBuster Master Mix.
  3. Optional: Add protease inhibitors. Protease inhibitors are compatible with BugBuster Master Mix.
  4. Incubate the cell suspension on a shaking platform or rotating mixer at a slow setting for 10–20 min at room temperature.
    Note: The extract should not be viscous after incubation.
  5. Remove insoluble cell debris by centrifugation at 16,000 × g for 20 min at 4°C. If desired, save the pellet for inclusion body purification.
  6. Transfer the supernatant to a fresh tube. For SDS-PAGE analysis, remove a small sample (25–50 μl) and combine with equal volume of 4X SDS Sample Buffer (Cat. No. 70607-3). Immediately heat for 3 min at 85°C to denature proteins and then store at –20°C until SDS-PAGE analysis.

Safety Considerations

Chemical/ Device/ Component Risk Risk Prevention
BugBuster Master Mix (MERCK, 2014) This item is not a hazardous substance and does not contain hazardous ingredients No prevention is needed for this product
Electrical Electrocution Do not touch unsealed or active wires, immediately report damaged wires. Turn off the power source when not in use or in the event of an emergency.
Acrylamide Toxic and carcinogenic. Toxic if swallowed, harmful contact with skin, skin and eye irritation. May cause genetic defects, cancer and damage to fertility. Wear appropriate PPE, including goggles, impermeable clothing and shoes and gloves. Control parameters ACGIH limit value 0.03 mg/m3 and OSHA PEL: 0.3mg/m3. A system of local or general exhaust is installed, if respiratory limits are exceeded respirators are required. (National Diagnostics, 2019)
Bis-Acrylamide Toxic and carcinogenic Toxic if swallowed, harmful contact with skin, skin and eye irritation. May cause genetic defects, cancer and damage to fertility. Wear appropriate PPE, including goggles, impermeable clothing and shoes and gloves. (ThermoFisher Scientific, 2010)
Ammonium Persulphate Oxidising, carcinogenic, irritant. May intensify fire, harmful if swallowed. Cause skin and eye irritation. May cause respiratory irritation and harmful to aquatic life Avoid dust generation, avoid contact with eyes, skin and clothing. Wear appropriate PPE including clothes, eye protection and lab clothing. (Global Safety Management Inc., 2015)

HSP Growth Assay (Preliminary Design)


  1. Prepare overnight culture of the control and target sample.
  2. From the overnight culture inoculate a day culture and allow it to grow up to 0.6 OD600.
  3. Induce protein production by adding IPTG.
  4. The OD600 of the culture is measured again before diluting the culture to an OD600 of 0.1 with new ampicillin-containing LB broth.
  5. Incubate the cultures in shaking incubators of 37° C and 45° C.
  6. Measure OD600 using the spectrophotometer every 30 minutes for 6 hours.

Safety Considerations

Chemical/ Device/ Component Risk Risk Prevention
Isopropyl β-D-1-thiogalactopyranoside (IPTG) Eye damage/eye irritation
Carcinogenic 		Wear PPE (safety glasses and gloves), wash skin thoroughly after handling If exposed to eyes, rinse with water for several minutes.
Heat Block High temperature Wear gloves when handling

HSP Survival Assay (Preliminary Design)


Procedure
  1. Prepare overnight culture of the control and target sample.
  2. From the overnight culture, inoculate a day culture and incubate at 37℃ until it reaches an OD600 of 0.4.
  3. Induce protein production with IPTG.
  4. Incubate the samples for 1 hr at 37℃ until OD600 is reached.
  5. Measure OD600 before heat shock.
  6. Set a temperature gradient of 37℃ to 61℃ in a PCR machine and allow it to run for 10 minutes.
  7. Dilute the samples with PBS if needed.
  8. Spread plate samples subjected to heat shock on antibiotics-containing agar plates.
  9. Incubate the plates at 37℃ overnight.
  10. Perform colony counting on the following day.

Safety Considerations

Chemical/ Device/ Component Risk Risk Prevention
SDS-PAGE: 30% acrylamide Skin/eye irritant
Carcinogenic
Reactive with oxidizing agents, acids, alkalis 		Wear PPE (safety glasses and gloves) Perform experiment in a fume hood
SDS-PAGE: tetramethylethylenediamine Corrosive
Highly flammable 		As above
SDS-PAGE: Ammonium persulfate Skin/eye irritant/sensitizer As above
Isopropyl β-D-1-thiogalactopyranoside (IPTG) Eye damage/eye irritation
Carcinogenic 		Wear PPE (safety glasses and gloves), wash skin thoroughly after handling If exposed to eyes, rinse with water for several minutes.

LB broth and LB agar preparation


LB Agar Recipe
  • 2% Agar
  • 1% NaCl
  • 1% tryptone
  • 0.5% yeast
LB Broth Recipe
  • 1% NaCl
  • 1% tryptone
  • 0.5% yeast
Procedure
Label flask with relevant details.
  1. Label flask with relevant details
  2. Weigh and add ingredients into the flask.
  3. Add in Mili-Q water. While adding the water, gently swirl the flask to aid with mixing.
  4. Send the prepared LB broth/agar for autoclave.

Safety Considerations

Chemical/ Device/ Component Risk Risk Prevention
Agar 20 g/L (G-Biosciences, 2017). Slightly harmful in contact with skin.
Fire hazard in finely divided state. Explosive with air in fine dust state or dust cloud.
Water pollutant (surface water). 		Wear gloves when using/ preparing LB agar. Wear safety goggles at all times and appropriate PPE. Keep container tightly closed. In case of dust production, wear dust mask with filter type P1. Eliminate fire sources/ naked flame. Keep away from fire/ sparks/ lighting/ heat/ ignition sources when using it. Store/ keep away from oxidizing agents. Keep the container in well-ventilated area. Avoid release to environment. Prevent spreading in sewers. Remove waste in accordance with local and/or national regulations. Remove to an authorized waste treatment plant.
NaCl 10 g/L (M-I Australia Pty Ltd, 2015). This product is not classified as hazardous.
Tryptone 10 g/L (SIGMA-ALDRICH, 2014) Incompatible with strong oxidizing agents.
Accidental release. 		Keep away from strong oxidizing agents. Keep container tightly closed in a dry and well-ventilated place. Do not let product enter drains.
Yeast extract 5 g/L (CDH, n.d.) Not a hazardous substance or mixture according to Regulation (EC) No. 1272/2008.

Overnight Culture


  1. Place 5 mL of LB broth into a labelled tube.
  2. Place 5 μL of Ampicillin stock solution into the same tube.
  3. Inoculate a single colony of bacteria from an agar plate into the tube.
  4. Incubate at 37°C overnight in a shaking incubator.

Safety Considerations

Chemical/ Device/ Component Risk Risk Prevention
Ampicillin trihydrate powder (ThermoFisher SCIENTIFIC, 2018). Acute toxicity, Repeated Dose Toxicity, Reproduction & Development Toxicity, Genetic Toxicity, Carcinogenicity (Carcinogen status: Group 3). Causes skin irritation, Causes serious eye irritation, May cause an allergic skin reaction, May cause allergy or asthma symptoms or breathing difficulties if inhaled. Wash face, hands and any exposed skin thoroughly after handling. Wear protective gloves/protective clothing/eye protection/face protection. Avoid breathing dust/fume/gas/mist/vapors/spray. In case of inadequate ventilation wear respiratory protection. Contaminated work clothing should not be allowed out of the workplace. Use only outdoors or in a well-ventilated area.
LB broth (SIGMA-ALDRICH, 2014) May be harmful if inhaled. May cause respiratory tract irritation. May be harmful if absorbed through skin. May cause skin irritation. May cause eye irritation. May be harmful if swallowed. Personal precautions: Use personal protective equipment. Avoid dust formation. Avoid breathing vapours, mist or gas. Ensure adequate ventilation. Avoid breathing dust. Environmental precautions: Do not let product enter drains. Methods and materials for containment and cleaning up: Pick up and arrange disposal without creating dust. Sweep up and shovel. Keep in suitable, closed containers for disposal.
E.coli expressing mCerulean and HSP22E Biological Hazard Treat as potentially hazardous. Wear personal protective equipment.

Streaking for Single Colonies


  1. Prepare antibiotic selection agar plates and label the plates.
  2. Collect sample of interest.
  3. Sterilize a 20μL pipette using 70% ethanol and paper towel.
  4. Set up a bunsen burner for sterile inoculation.
  5. Using a 20μL pipette, scrape some cells from the sample.
  6. Inoculate bacterial cells from sample of interest onto the agar plate.
  7. Streak out the culture, swapping pipette tips between streaks to maintain sterility.
  8. Incubate at 37°C overnight.

Safety Considerations

Chemical/ Device/ Component Risk Risk Prevention
Bunsen Burner Fire hazard, burn risk Keep flammable materials away from the flame. Secure all loose hair and clothing. Ensure the gas line is secure.

References

CDH YEAST EXTRACT POWDER - MATERIAL SAFETY DATA SHEET SDS/MSDS. Available at: https://www.cdhfinechemical.com/images/product/msds/47_1250660188_YEASTEXTRACTPOWDERMSDS.pdf (Accessed: 1st September 2021).

G-BIOSCIENCES. 2017. Agar (Bacteriological) Safety Data Sheet. Available at: https://www.gbiosciences.com/image/pdfs/msds/BTNM-0012_msds.pdf (Accessed: 1st September 2021).

GLOBAL SAFETY MANAGEMENT INC. 2015. Ammonium Persulfate ACS. Available: https://bionium.miami.edu/_assets/pdf/msds/ammonium-persulfate.pdf.

MECRK. 2008. BugBuster® Protein Extraction Reagent.

MERCK. 2014. Safety Data Sheet for BugBuster® Master Mix 71456. Available: https://www.merckmillipore.com/AU/en/product/msds/EMD_BIO-71456?Origin=PDP

MERCK. 2017. SAFETY DATA SHEET. Available at: https://www.merckmillipore.com/AU/en/product/msds/MDA_CHEM-137041?Origin=PDP (Accessed: 1st September 2021).

M-I AUSTRALIA PTY LTD. 2015. Safety Data Sheet SODIUM CHLORIDE POWDER (SALT PVD or GRANULAR SALT). Available at: https://www.originenergy.com.au/content/dam/aplng/compliance/msds/SODIUM%20CHLORIDE%20SDS.pdf (Accessed: 1st September 2021).

NATIONAL DIAGNOSTICS. 2019. Safety data sheet: Acrylamide, EC-201. Available: http://www.nationaldiagnostics.com/msds_pdfs/sds_product10.php?cat_num=EC-201

SCIENTIFIC, T. 2018. SAFETY DATA SHEET 1-7. Available at: https://www.fishersci.com/store/msds?partNumber=AC612290250&productDescription=AMPICILLIN+TRIHYDRATE+25GR&vendorId=VN00033901&countryCode=US&language=en (Accessed: 1st September 2021).

SIGMA-ALDRICH. 2014. L-(-)-tryptophan - Safety Data Sheet. Available at: https://www.nwmissouri.edu/naturalsciences/sds/l/L-Tryptophan.pdf (Accessed: 1st September 2021).

THERMOFISHER SCIENTIFIC. 2010. Safety Data sheet: Bis-Acrlamide (Electrophoresis). Available: https://www.fishersci.com/store/msds?partNumber=BP17125&productDescription=BIS-ACRYLAMIDE+25+GM&vendorId=VN00033897&countryCode=US&language=en