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Experiments

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Starter pack

70% Ethanol Protocol (starter pack)

Introduction

Ethanol is a commonly used antiseptic in the lab environment. It has the highest effective concentration at 70% compared to a stronger solution. This occurs due to alcohol’s ability to coagulate protein on contact. A higher concentration of ethanol inflicts a very rapid coagulation of protein in the cell wall or membrane of target organism, effectively blocking further penetration of the organism and its subsequent neutralization. Because of this, a more diluted 70% concentration is used for optimal penetration.

Safety precautions

SDS (safety data sheet): Refer to the SDS sheets for all listed materials before entering the lab. Be prepared to answer any questions regarding th3e information on these sheets.

PPE (Personal protective equipment): Proper lab attire should be worn throughout the experiment: This means that upon entering the lab you should be wearing long pants and close-toed shoes. Contact lenses should not be worn. Furthermore, a lab coat, goggles, and gloves should be worn at all times, and long hair should be tied back.

Hazards: Highly flammable, irritant

Materials

Reagents

• 95% ethanol
• Distilled water

Equipment

• Wash bottle
• Graduated cylinder

Procedure

This protocol results in 200mL of stock solution.

1. Obtain a graduated cylinder with 42.1mL dH₂O (distilled water).
2. Obtain a graduated cylinder with 157.9mL ethanol.
3. Transfer 42.1mL dH₂O into a wash bottle.
4. Transfer 157.9mL ethanol into the wash bottle, minimizing any splashing of the solution.
5. Close the wash bottle and swirl it in circular motion to mix

Leaving the lab

• Clean dirty glassware, or at least set aside the glassware to be cleaned by a designated individual.
• Wipe down your workspace.
• Ensure that all materials have been returned to their places, and that the plates have been properly stored in the fridge.
• Dispose of all disposable tubes and pipette tips used in biohazard containers.
• Make sure your plates are labelled and put in a place they can be found.

Acknowledgements

Made by Katariina Jaenes (iGEM Toronto 2015).

Protocols of previous iGEM teams were used to make this guideline.

Antibacterial Stock Preparation (starter pack)

Introduction

The preparation of antibiotic stock is a relatively simple series of mixing and dilutions. The antibiotic stocks are typically made from four antibiotics: Ampicillin, Kanamycin, Tetracycline and Chloramphenicol. With the exception of chloramphenicol, the other antibiotics are light sensitive and once stock is prepared they are wrapped in foil to reduce light exposure.

An antibiotic comes in a powder form as the basic stock and is mixed with milli-Q water to form a stock solution. Antibiotic stock preparation follows the same basic process for each antibiotic, varying only in concentration which is dependent on the type of antibiotic used.

Antibiotic stocks are prepared for use either in liquid or solid media. In the case of liquid media, the antibiotic stock is simply added and mixed in. For solid media, usually agar and LB, the media is sent for autoclaving, and while the media is still hot the antibiotic is added and mixed. This is to ensure that the antibiotic can be mixed evenly amongst the media before it solidifies into a solid state.

Antibiotics are used for the purpose of selection. In terms of synthetic biology, plasmids confer selective antibiotic resistance when successfully transformed into their target bacteria. Using liquid or solid growth media that has been treated with antibiotics provides a selection control for those bacteria that have successfully incorporated the plasmid.

Basic Terminology and Concepts

There are some terms which will be commonly used to describe the preparation of antibiotic stocks. Familiarity and understanding of these terms is key to comprehend the protocol.

• Milli-Q: Water that has been purified through successive steps of filtration and deionization. The standard used in our lab is typically 18.2MΩ·cm at 25°C, measured in resistance due to the lack of ions. The filters used are 0.22μm in size to ensure a high level of purity. This water is used for preparing our antibacterial stock to ensure purity.
• LB (Lysogeny Broth): a very standard and simple media to create due to its recipe consisting of 3 components - tryptone, yeast extract, and sodium chloride. This mix of anhydrous ingredients is added to water and then autoclaved, producing liquid media. The production of solid LB for plates is done by adding agar, a protein isolated from certain species of seaweed which coagulates the liquid into a gel-like form when cooled. This is done prior to autoclaving and is poured into the plates while still hot, where it will cool into the plate shape.
• Aliquot: An aliquot is a term to denote a certain quantity of something. In this case an aliquot of antibiotic stock would denote either 1mL or 0.5mL, depending on the antibiotic.
• Antibiotic: Compounds which inhibit bacterial growth. They act either bacteriostatically by preventing reproduction of the bacteria, or bacteriocidally where they directly kill the bacteria. Generally bacteriocides work by interfering with the synthesis of peptidoglycan in the bacteria’s cell walls. Tetracycline is an example of a bacteriostatic, where it acts by binding to the ribosomes of prokaryotic bacteria and inhibits translation.
• Autoclave: A piece of equipment used for sterilization. The autoclave performs much like a pressure cooker: it subjects the contents inside it to a high temperature and high pressure steam bath. Usually the temperature is 121°C and at 15lbs/in², 20 minutes is enough to kill most microorganisms and render equipment sterile. When adding antibiotics to media, it is done after autoclaving so that the heat does not destroy the antibiotic activity.

Safety precautions

SDS (safety data sheet): Refer to the SDS sheets for all listed materials before entering the lab. Be prepared to answer any questions regarding th3e information on these sheets.

PPE (Personal protective equipment): Proper lab attire should be worn throughout the experiment: This means that upon entering the lab you should be wearing long pants and close-toed shoes. Contact lenses should not be worn. Furthermore, a lab coat, goggles, and gloves should be worn at all times, and long hair should be tied back.

Hazards: Carcinogen (D2A), mutagen (D2B), Acute oral toxicity

Materials

Reagents

• MilliQ water
• 4g Ampicillin
• 800mg Kanamycin
• 400mg Tetracycline
• 2.72g Chloramphenicol
• 80mL 70% EtOH
• 80mL 100% EtOH

Equipment

• Antistatic weighing boat
• 100mL Pyrex bottle
• Magnetic stir bar
• Magnetic stirrer
• Analytical balance
• Milli-Q water dispenser
• 50mL Falcon tubes
• Aluminum foil
• 20mL syringe
• 0.22μm Filter
• 1mL microcentrifuge tubes

Ampicillin

Notes

Stocks and Usage:

• Stock concentration 50mg/mL in milliQ water
• Aliquots: 500μL (use a P1000 set to 0500)
• Working Concentration: 50μg/mL preparation of 80mL stock solution

Ampicillin is kept in the 4°C fridge, and is light sensitive. To ensure your stock solution is not degraded, cover all microcentrifuge tubes used for storing the solution with foil (you could use a falcon tube and wrap it with foil as well).

Procedure

1. Weigh 4g ampicillin onto an antistatic weighing boat.
2. Add 80mL milliQ water to a 100mL Pyrex bottle.
3. Add the ampicillin to the milliQ water.
4. Place a small magnetic stir bar into the solution and place the Pyrex bottle on the stirrer. Set at 300-600rpm and stir until dissolved.
5. Filter sterilise the solution into 50mL Falcon tubes using a 20mL syringe outfitted with a 0.22μm filter.
6. Aliquot into the appropriate microcentrifuge tubes, labelled with an “A” on top, and store in the Nalgene racks found in the 20°C fridge

Kanamycin (KAN)

Notes

Stocks and Usage:

• Stock Concentration 10mg/mL in milliQ water
• Aliquots: 1mL (use a P1000 set to 1000)
• Working Concentration: 50μg/mL preparation of 80mL stock solution
• Add 5mL of stock per litre of LB

Kanamycin is kept in the 4°C fridge, and is light sensitive. To ensure your stock solution is not degraded, cover all microcentrifuge tubes used for storing the solution with foil.

Procedure

1. Weigh 800mg kanamycin onto an antistatic weighing boat.
2. Add 80mL milliQ water to a 100mL Pyrex bottle.
3. Add the kanamycin to the milliQ.
4. Place a small magnetic stir bar into the solution and place the Pyrex bottle on the stirrer. Set at 300-600rpm and stir until dissolved.
5. Filter sterilise the solution into 50mL Falcon tubes using a 20mL syringe outfitted with a 0.22μm filter
6. Aliquot (1mL) into the appropriate microcentrifuge tubes, labelled with a “K” on top. Store in the Nalgene racks found in the 20°C fridge. (Make sure microcentrifuge tubes are covered with foil)

Tetracycline

Notes

Stocks and Usage:

• Stock concentration 5mg/mL in 70% EtOH (N.B. 70%, not 100% EtOH!)
• Aliquots: 1mL (use a P1000 set to 1000)
• Working Concentration: 20μg/mL preparation of 80mL stock solution

Tetracycline is kept in the 4°C fridge, and is light sensitive. To ensure your stock solution is not degraded, cover all microcentrifuge tubes used for storing the solution with foil.

Procedure

1. Weigh 400mg tetracycline onto an antistatic weighing boat.
2. Add 80mL 70% EtOH to a 100 mL Pyrex bottle.
3. Add the tetracycline to the 70% EtOH.
4. Place a small magnetic stir bar into the solution and place the Pyrex bottle on the stirrer. Set at 300-600rpm and stir until dissolved.
5. Recommended, but optional, because of storage in 70% EtOH: Filter sterilise the solution into 50mL Falcon tubes using a 20mL syringe outfitted with a 0.2μm filter.
6. Aliquot into the appropriate microcentrifuge tubes, labelled with a “T” on top. Store in the Nalgene racks found in the 20°C fridge.

Chloramphenicol (CAM)

Notes

Stocks and Usage:

• Stock Concentration 34mg/mL in 100% EtOH (N.B. 100%, not 70%!)
• Aliquots: 1mL (P1000 set to 1000)
• Working Concentration: 25μL/mL preparation of 80mL stock solution

Chloramphenicol is kept with the general chemicals, and is not light sensitive. The microcentrifuge tubes do not need to be covered with foil to store chloramphenicol.

Procedure

1. Weigh 2.72g chloramphenicol onto an antistatic weighing boat.
2. Add 80mL 100% EtOH to a 100mL Pyrex bottle.
3. Add the chloramphenicol to the 100% EtOH.
4. Place a small magnetic stir bar into the solution and place the Pyrex bottle on the stirrer. Set at 300-600rpm and stir until dissolved.
5. Aliquot (1mL) into the appropriate microcentrifuge tubes, labelled with a “C” on top. Store in the Nalgene racks found in the 20°C fridge.

Note: No filter sterilization is needed because it is stored in 100% EtOH.

Leaving the Lab

Prior to leaving the lab, you should:

• Clean dirty glassware, or at least set aside the glassware to be cleaned by a designated individual.
• Wipe down your workspace.
• Ensure that all materials have been returned to their places, and that the plates have been properly stored in the fridge.

Acknowledgements

Protocols of previous iGEM teams were used to make this guideline.

Kayla’s M9 Media Preparation (starter pack)

Introduction

Modified M9 media is used for Escherichia coli cultures. It consists of an additional nitrogen source that seems to help with enzyme expression of heterologous pathways. The following recipe uses 3% of final glucose concentration but this can be modified accordingly.

Materials

The following components need to be autoclaved/filter sterilized separately. When sterilizing components by filtration, ensure that you filter into a sterile (autoclaved) bottle or container.

• 5X Modified M9 Salts (per Litre):
  • 64g Na₂HPO₄•7H₂O (or 33.89g Na₂HPO₄ Anhydrous)
  • 15g KH₂PO₄
  • 2.5g NaCl
  • 10.0g NH₄Cl
  • 5.0g (NH₄)₂SO₄
• 100mL of 1M MgSO₄:
  • 24.65g MgSO₄•7H₂O
  • Water
• 100mL of 1M CaCl₂:
  • 14.70g CaCl₂•2H₂O
  • Water
• 1L of (10X) 1M MOPS:
  • 209.3g MOPS
  • Deionized water
  • NaOH
• Trace Metals (1000X) (per litre of stock):
  • 1.6g FeCl₃
  • 0.2g CoCl₂•6H₂O
  • 0.1g CuCl₂
  • 0.2g ZnCl₂•4H₂O
  • 0.2g NaMoO₂
  • 0.05g H₃BO₃
• 50% Glucose:
  • 500g D-glucose
  • Deionized water

Procedure

5X Modified M9 Salts

1. Combine 64g Na₂HPO₄•7H₂O (or 33.89g Na₂HPO₄ Anhydrous), 15g KH₂PO₄, 2.5g NaCl, 10.0g NH₄Cl, and 5.0g (NH₄)₂SO₄ per litre of stock
2. Autoclave or filter sterilize

100mL of 1M MgSO₄

1. Add 24.65 g of MgSO₄•7H₂O to 87mL water.
2. Make up volume to 100mL.
3. Autoclave.

100mL of 1M CaCl₂

1. Add 14.70g CaCl₂•2H₂O to 94.5mL water.
2. Make up volume to 100mL.
3. Autoclave.

1L of (10X) 1M MOPS

1. Add 209.3g of MOPS (free acid) to 800mL deionized water.
2. Adjust to pH 7 with NaOH.
3. When dissolved make up volume to 1L.
4. Filter through 0.22uM filter or can be autoclaved.

Trace Metals (1000X)

1. Prepare trace metal stock in 0.1 M HCl
2. Add 1.6g FeCl₃, 0.2g CoCl₂•6H₂O, 0.1g CuCl₂, 0.2g ZnCl₂•4H₂O, 0.2g NaMoO₂, and 0.05g H₃BO₃ per litre of stock desired

50% Glucose

1. Add 500g D-glucose to 500mL deionized water.

• NOTE: You have to add glucose slowly to the water while mixing and heating, otherwise glucose will not fully dissolve.

2. Autoclave to sterilize.

Preparation of final modified M9 media

Note that in our experiment, we will be making M9 supplemented with Arabinose, and thus will be making a 10% stock which we will dilute further to 0.2% for all assays.

Component (stock concentration)	Amount to add per 1L stock (mL)	Amount to add per 500mL stock (mL)	Amount to add per 250mL stock (mL)	Amount to add per 200mL stock (mL)	Amount to add per 100mL stock (mL)
5X Modified M9 salts	200	100	50	40	20
Glucose (50%)	60	30	15	12	6
MgSO<sub>4</sub> (1M)	1	0.5	0.25	0.2	0.1
CaCl<sub>2</sub> (1M)	0.1	0.05	0.025	0.02	0.01
Thiamine (0.05mg/mL)	1	0.5	0.25	0.2	0.1
MOPS (1M)	100	50	25	20	10
Trace Metals (1000X)	1	0.5	0.25	0.2	0.1
Deionized water (with MOPS/without MOPS)	636.9/736.9	318.5/368.5	159.3/184.3	127.4/147.4	63.7/73.7

1. Add the components listed in the table above in the approrpiate amounts.
2. Thiamine is light and temperature sensitive; therefore, store the stock at 4°C. Trace metals are light sensitive; therefore, store the stock in an amber bottle. Once thiamine and trace metals are added to the media, store the media at 4°C.

Note: Supplement with required antibiotic before use.

Acknowledgements

1000X Trace Metals: Refer to Causey et al. (2003) PNAS

LB media preperation (starter pack)

Introduction

Note: This protocol makes 500mL of broth or ~25 plates.

In order for bacteria to be successfully cultured, they must be grown in the appropriate media. LB, also known as Lysogeny broth, is a nutrient rich broth that is a standard for culturing Escherichia coli, as it allows for quick growth and high yields. Therefore, the proper preparation of LB will be crucial to maintaining our bacterial stock throughout the summer. Furthermore, addition of agar to LB broth creates a gel for bacteria to grow upon, and is therefore used for plating bacterial cultures on petri dishes.

** WATCH THESE VIDEOS BEFORE ATTEMPTING THIS PROTOCOL**

• Making LB agar: https://www.youtube.com/watch?v=74Ndr9AVowM
• Pouring plates: https://www.youtube.com/watch?v=X4L16Vu5MSk

Basic Terminology and Concepts

• Agar vs. Agarose: Agar is used for making petri plates to culture organisms, while agarose is used for making gels, in the likes of SDS-PAGE and gel electrophoresis

Safety Precautions

SDS (safety data sheet): Refer to the SDS sheets for all listed materials before entering the lab. Be prepared to answer any questions regarding the information on these sheets.
PPE (Personal protective equipment): Proper lab attire should be worn throughout the experiment. This means that upon entering the lab you should be wearing long pants and close-toed shoes. Contact lenses should not be worn. Furthermore, a lab coat, goggles, and gloves should be worn at all times, and long hair should be tied back.
Autoclave: The autoclave should only be handled by leads and managers. Note that any autoclaved materials may still be hot and should therefore be handled with caution. Be careful not to burn yourself.

Materials

Reagents

• 5g Bacto-tryptone
• 2.5g yeast extract
• 5 gNaCl
• 7.5g agar (Only necessary if making LB agar plates)
• 500mL of dH₂O (distilled water)

Equipment

• 1L Pyrex bottle
• 1L graduated cylinder
• Filter paper and scoopula
• Stack of sterile plates (this protocol makes approximately 25)
• Bunsen burner/ethanol burner
• 70% EtOH wash bottle
• Paper towels/wipes

Procedure

Part 1: Making the LB broth

This part can be carried out at a regular lab bench.

1. Obtain a clean 1L pyrex bottle
2. Obtain a graduated cylinder with 500mL of dH₂O and add to the bottle. Record the amount added.
3. Using filter paper, separately measure out 5g of NaCl, 5g of Tryptone, and 2.5g of yeast extract on a scale and add them to the bottle. Swirl the bottle in a circular motion to mix. Remember to recalibrate your scales in between measurements.
4. If you are making LB agar plates, weigh and add 7.5g of agar and swirl to mix. Record the amount added.

Note the contents do not necessarily need to be completely in solution before autoclaving.

Part 2: Autoclaving

1. Lightly seal the top of the beaker with aluminium foil, and label the beaker with autoclave tape stating LB (agar)–[your name]–[date]–[media number]–iGEM.

Unless you have been trained to use the autoclave, you will not be conducting the following steps in this part

2. Use appropriate transportation protocols to bring the LB bottle into the autoclave room. Remember to store the beaker in an autoclavable basin, in case of spills.
3. Check the water level on the autoclave, if necessary. Autoclave on the liquid setting for approximately 20 min.

• The contents of the beaker will be hot after autoclaving, therefore take the necessary measures to prevent burns.

4. After autoclaving, allow the LB media to cool to 55°C before handling.
   • Use laser thermometer to check the temperature of the glass.
5. The LB broth can be stored in sterile conditions at room temperature, and should be good for 3-4 months. Flame the lip of the bottle each time the LB is used. If the LB contains antibiotics, store in a -4°C freezer.
   • However, it is not recommended to store LB with antibiotics as the antibiotics will degrade over time

Part 3: Pouring the plates (for LB agar)

While pouring the plates, it is crucial to maintain a sterile environment. This should be done in room WB 303, with a sterile environment provided by a lit Bunsen burner.

Note: steps 1-3, in addition to the clean up from Part 1, can be done while waiting for autoclave.

1. Sterilize the workspace with 70% EtOH before depositing your materials. Light the Bunsen burner.
2. Obtain a stack/roll of empty plates. The plates should still be in their plastic sleeve/wrapping, as they should be sterile. Don’t throw out the wrapping as it can be used to store the plates. It is essential that you minimize any chance of contaminating the plates. Make sure that you open the package at the top and expose the plates as minimally as possible.
   • Note that this protocol makes ~25 plates.
3. Once you take the plates out, store them upside down on your lab bench. Label the plates with [your name]–iGEM 2017–[date]–[media number]–[antibiotic stripe]. Once labelled, you may stack the plates to free up workspace.
   • One stripe along the sides corresponds to CAM, two stripes corresponds to AMP
4. Allow the LB media to cool before pouring. The LB will start to settle at ~30°C.
5. If you are preparing selective media, add antibiotic to the mixture. Swirl the flask in a circular motion to mix. If you don’t know whether or not you are preparing selective media, ASK.
   • Use concentrated liquid stocks for the antibiotics.

• Recommenced antibiotic concentrations:
  • Chloramphenicol (CAM): 25μg/mL
  • Ampicillin (AMP): 100μg/mL

6. Take an empty plate and open it slightly. You do not need to open it all the way to pour the agar.
7. Pour agar until 2/3 of the plate has been covered, or approximately half of the plate has been filled when viewed from the side. Pour the agar slowly to prevent the formation of bubbles. Swirl the plate in a circular motion to distribute the media evenly on the plate.

• If you pour too much LB, you will not be able to produce 25 plates. If you don’t pour enough media, it may minimize bacterial growth.

8. After pouring, set the plates to cool in stacks of 4-5 to save space and flip the plates to prevent condensation forming on the agar. Don’t stack plates too high - we want to minimize the risk of spills. Allow the plates to cool for at least 20 minutes until the agar has solidified.
9. Rinse the Pyrex bottle with water before the remnants solidify and become hard to remove.
10. The plates can then be stacked and stored in plastic bags (ideally, re-use the plastic bags that the plates came in.)
11. Store LB agar plates in a 4°C freezer. They should be good for 1-2 months.

Leaving the lab

Prior to leaving the lab, you should:

• Clean dirty glassware, or at least set aside the glassware to be cleaned by a designated individual.
• Wipe down your workspace.
• Ensure that all materials have been returned to their places, and that the plates have been properly stored in the fridge.

Acknowledgements

• Addgene: Protocol – Making LB Agar Plates for Bacteria. From: https://www.addgene.org/plasmid-protocols/bacterial-plates
• Department of Ecology and Evolutionary Biology, UCLA. Making LB Agar Plates. From: https://www.eeb.ucla.edu/Faculty/Barber/Web Protocols/LB Agar Plates.pdf
• iGEM Toronto Luria Broth (LB) protocol
• Sezonov G, Joseleau-Petit D, D’Ari R. Escherichia coli Physiology in Luria-Bertani Broth. Journal of Bacteriology. 2007;189(23):8746-8749. doi:10.1128/JB.01368-07.

SOC Medium Protocol (starter pack)

Introduction

SOC is a variant of the rich media SOB (super optimal broth) with catabolite repression. This means that glucose is supplemented in the media, allowing for optimal metabolic conditions for the bacteria. SOC increases the transformation efficiency of cells, as it provides ample nutrients to cells that have recently undergone stress as result of having been made competent. Accordingly, it will be used in bacterial transformation to stabilize the cells and to increase transformation yields. Since SOC is high in nutrients, it is more easily contaminated than LB or King’s B media.

Safety Precautions

SDS (safety data sheet): Refer to the SDS sheets for all listed materials before entering the lab. Be prepared to answer any questions regarding the information on these sheets.

PPE (Personal protective equipment): Proper lab attire should be worn throughout the experiment: This means that upon entering the lab you should be wearing long pants and close-toed shoes. Contact lenses should not be worn. Furthermore, a lab coat, goggles, and gloves should be worn at all times, and long hair should be tied back.

Autoclave: The autoclave should only be handled by execs. Note that any autoclaved materials may still be hot and should therefore be handled with caution. Be careful not to burn yourself.

Materials

Reagents

• 1.802g glucose
• 10g tryptone
• 2.5g Yeast extract
• 0.584g NaCl
• 0.093g KCl
• 1.016g MgCl₂ (anhydrous)
• 1.234g MgSO₄•7H₂O
• 500mL dH₂O (distilled water)

Equipment

• 2×1L pyrex bottle (must have cap)
  • Note: A smaller pyrex bottle may be used to accommodate the glucose solution
• 1L graduated cylinder
• Weighing boats and scoopula
• 70% EtOH wash bottle
• Paper towels/wipes

Procedure

Part 1: Making the SOC broth

This step can be carried out at a regular lab bench.

1. Obtain two 1L pyrex bottles. Ensure that the bottles can be sealed with a cap - this will help prevent contamination and enable long term storage.
2. Obtain a graduated cylinder with 500mL dH₂O (distilled water).
3. Using filter paper, separately measure out 10g tryptone, 2.5g yeast extract, 0.292g NaCl, 0.093g KCl, 1.016g MgCl₂ anhydrate, and 1.234g MgSO₄•7H₂O on a scale and add them to the 1L bottle.
   • Remember to recalibrate your scales in between measurements.
4. Add 400mL dH₂O. Swirl the flask in a circular motion to mix.
5. In the separate bottle, and measure out 1.802g glucose on a scale and add the rest of the 100 mL dH₂0. Recalibrate your scales in between measurements.

Note: The 1 L bottles are autoclaved separately, as the contents will react if autoclaved together.

Autoclaving

ONLY DONE BY EXECS

1. Lightly seal the top of the bottles with aluminium foil, or unscrew the caps. Label both with autoclave tape. Include a label with SOC – [your name] – [date] - iGEM 2017.

• **Unless you have been trained to use the autoclave, you will not be conducting the following steps 2-4. **

2. Use appropriate transportation protocols to bring the bottles into the autoclave room. Remember to store the beaker in an autoclavable basin, in case of spills.
3. Check the water level on the autoclave, if necessary. Autoclave on the liquid setting for approximately 20 min.
4. The contents of the bottles will be hot after autoclaving, so take necessary measures to prevent burns. After autoclaving, allow the media too cool to 55°C before handling.

Part 2: Making the SOC broth

This part should be done in room WB303, in sterile conditions close to a Bunsen burner.

1. In a sterile environment, slowly add the autoclaved 1.802g glucose and dH₂O to the beaker containing the autoclaved 1.016g MgCl₂ and 1.234 g MgSO₄, 10g tryptone, 2.5g yeast extract, 0.292g NaCl, 0.093g KCl, and dH₂0.

• Flame the lip of the bottle before transferring the contents. Swirl to mix, and seal tightly to prevent contamination. Flame the cap before sealing.

2. The SOC broth can be stored in sterile conditions at room temperature, and should be good for a 3-4 months. Flame the lip of the bottle each time the SOC is used. SOC should be handled carefully, as it is especially prone to contamination.

Before leaving the lab

• Clean dirty glassware, or at least set aside the glassware to be cleaned by a designated individual.
• Wipe down your workspace.
• Ensure that all materials have been returned to their places, and that the plates have been properly stored in the fridge.

Acknowledgements

• Cold Spring Harbour Protocols. SOC Medium for E.coli. From: http://cshprotocols.cshlp.org/content/2012/6/pdb.rec069732.short
• Sun QY et al. Culture of Escherichia coli in SOC medium improves the cloning efficiency of toxic protein genes. Anal Biochem. 2009: From http://www.ncbi.nlm.nih.gov/pubmed/19622338
• Qiagen. Recipe for SOC Medium. From: https://www.qiagen.com/ca/resources/faq?id=d3be05d0-ec02-4ced-aabe-dcd2a1061920&lang=en
• Protocol by Katariina Jaenes