Team:Manchester/Project/Protocols

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Medium and Buffers

20% (w/v) glucose solution

1. Prepare the solution by mixing the ingredients stated above.
2. Sterilize in an autoclave before using it to prepare the SOC medium.

50% (w/v) glycerol solution

1. Prepare the solution by mixing the ingredients stated above.
2. Sterilize in an autoclave.

*Source from 2015 iGEM Exeter

Electrophoresis buffers

For nucleic acids DNA/RNA separation

• TAE buffer (Tris-acetate-EDTA)
• TBE buffer (Tris-borate-EDTA)
• LAB buffer (Lithium-acetate-borate)

1. Prepare the solution by mixing the ingredients stated above.

LB agar (Luria-Bertani agar)

If not using pre-mixed LB agar powder, prepare the materials as below:

1. In a 1L Erlenmeyer flask, swirl and mix the solution.
2. Cover the top of the flask with a lid/aluminum foil and label with autoclave tape.
3. Autoclave the liquid setting for 20 minutes or according to your autoclave's specifications.
4. After removing the solution from the autoclave, allow the agar solution to cool to 55°C in an oven or water bath.
5. When pouring the LB agar into plates, keep the bench area sterile by working near a flame or Bunsen burner. Alternatively, prepare the plates in a vacuum hood.
6. Add the appropriate amount of desired antibiotic (refer the table below) to the solution and swirl to mix.
7. Pour approximately 20mL of LB agar per 10cm polystyrene Petri dish.
8. Place the lids on the plates and allow them to cool for until the agar is solidified.
9. Label the bottom of plates with antibiotic and date before storing in plastic bags or sealed with Parafilm at 4°C.

Table source from New England Biolabs

Additional note:

• Antibiotic carbenicillin can be substituted for ampicillin in antibiotic selection plates ^[1].

Source from

LB medium (Luria-Bertani medium)

If not using pre-mixed LB broth powder, prepare the materials as below:

1. Prepare the solution by mixing the ingredients stated above.
2. Sterilize in an autoclave.

SOB medium (Super Optimal Broth)

1. Prepare the solution by mixing the ingredients stated above.
2. Sterilize in an autoclave before using it to prepare SOC medium.

Additional notes

• Some formulations of SOB use 10 mM MgCl₂ and 10 mM MgSO₄ instead of 20mM MgSO₄.
• SOB medium is also available dry pre-mixed from Difco, 0443-17
• Adjust to pH 7.5 prior to use. This requires approximately 25mL of 1M NaOH per liter.

Source from OpenWetWare

SOC medium (Super Optimal broth with Catabolite repression)

1. Prepare the solution by mixing the ingredients stated above.

*Source from OpenWetWare

TSS buffer (Transformation & Storage Solution)

1. Prepare the solution by mixing the ingredients stated above.
2. Filter sterilize using a 0.22 μm filter.
3. Store at 4°C or -20°C.

*Source from OpenWetWare: TSS

Transformation

Electrocompetent cells

Ingredients:

• LB media
• 50% (w/v) glycerol solution

Before starting the procedure, be sure to:

• Pre-chill centrifuge and cool rotor to 4°C
• Pre-chill 1L of 10% (w/v) glycerol solution on ice
• Pre-chill 500mL/1000mL Falcon tubes on ice

During the procedure:
Work under flame at all times.

1. Grow a 5mL overnight culture of cells in LB media.
2. In the morning, dilute this culture by at least 1/100 into a 50mL of fresh LB media in a 200mL conical flask.
3. Incubate them at 37°C.
4. Monitor growth of the cells every 30 minutes by measuring OD at 600nm (OD600) by filling up 750 µL of culture into cuvette. Use LB medium as blank.
5. Once the cells are ready at OD600 = 0.4-0.6, harvest the cells to prepare for electroporation.
6. Place cultures on ice for 15 minutes.



All subsequent steps should be carried out at 4°C.
Cells should be kept on ice wherever possible.



7. Pour each 250mL culture into pre-chilled 500mL (or 1000mL) Falcon tubes.
8. Centrifuge at 5000rpm for 10 minutes.
9. Pour off the supernatant and aspirate any residual broth.
10. Add 250 mL of pre-chilled 10% (w/v) glycerol to each of the centrifuge bottles and completely suspend the cells by pipetting the solution up and down.
11. Centrifuge at 5000rpm for 10 minutes.
12. Pour off the supernatant.
13. Completely suspend the cells in 250mL glycerol and re-centrifuge at 5000rpm for 10 minutes.
14. Pour off the supernatant and suspend the cells in the residual glycerol by pipetting up and down.
15. At this point you can electroporate or freeze the cells away. To freeze, add 100µL of the culture to microcentrifuge tubes on ice.
16. Once you have used all of the culture, transfer the tubes to dry ice for 10 minutes.
17. Once the cultures are frozen, transfer them to a -80°C freezer. The cultures should be good for more than 6 months.

Electroporation

Ingredients:

• SOC medium
• LB agar plates

Before starting the procedure, be sure to:

• warm SOC medium to 37°C

1. Add 50µL of competent cells + 1 µL of DNA. Mix well and place on ice for 5 minutes.
2. Transfer mix to 0.1cm electroporation cuvette (BioRad, #1652083)
3. Perform electroporation in a MicroPulse Electroporator (BioRad) using one pulse of 1.80 kV (EC1).
4. Measure the time constant (~5ms).
5. Add 950µL of pre-warmed SOC medium (37°C) to the cuvette immediately after electroporation.
6. Transfer to a fresh sterile 2mL tubes.
7. Incubate at 37°C for 2 hours.
8. Plate 50, 100, 200 µL (appropriate dilutions) and the rest of the cells (by centrifuging for 1 sec then decanting the liquid and resuspending with what is left in the tube) into LB agar plates using a plastic spreader.
9. Incubate at 37°C overnight (approximately 16-18 hours).
10. Look for transformants the next morning.
11. Take note of the dilutions made to use for colony calculations next day.

*Source from New England Biolabs

Chemical competent cells

Ingredients:

• LB media
• TSS buffer

Before starting the procedure, be sure to:

• Pre-chill 1mL Eppendorf tubes
• Pre-chill TSS buffer
• Pre-chill Falcon tubes

During the procedure:
Work under flame at all times.

1. Grow a 5mL overnight culture of cells in LB media.
2. In the morning, dilute this culture by at least 1/100 into a 50mL of fresh LB media in a 200mL conical flask.
3. Incubate them at 37°C.
4. Monitor growth of the cells every 30 minutes by measuring OD at 600nm (OD600) by filling up 750 µL of culture into cuvette. Use LB medium as blank.
5. Once the cells are ready at OD600 = 0.2-0.5, harvest the cells to prepare for chemical transformation.
6. Split the culture into 50mL Falcon tubes and incubate on ice for 10 minutes.



All subsequent steps should be carried out at 4°C.
Cells should be kept on ice wherever possible.



7. Centrifuge for 10 minutes at 3000 rpm and 4°C.
8. Remove the supernatant. Pipette out any remaining media.
9. Re-suspend in pre-chilled TSS buffer. The volume of TSS to use is 10% of the culture volume (i.e 25mL culture = 2.5mL of TSS to be added) that you spun down.

You may need to vortex gently to fully re-suspend the culture, keep an eye out for small cell aggregates even after the pellet is completely off the wall.

10. Add 100µL aliquots to your chilled 1mL Eppendorf tubes and store at -80°C.



*Source from OpenWetWare

Chemical transformation

Ingredients:

• SOC medium
• LB agar plates

Before starting the procedure, be sure to:

• Thaw competent cells from -80°C on ice

During the procedure:
Work under flame at all times.

1. Add 50µL of competent cells + 1 µL of DNA (2.5µL of DNA if doing ligation).
2. Mix well and place on ice for 30 minutes, or 5-10 minutes if in a rush.
3. Heat shock the cells at 42°C for 30 seconds.
4. Incubate on ice for 2 minutes.
5. Add 450µL of SOC medium (kept in 4°C fridge) to the cells.
6. Incubate for 45 minutes at 37°C in the shaker (30 minutes for Carbenicillin).
7. Plate 50, 100, 200µL (appropriate dilutions) and the rest of the cells (by centrifuging for 1 second then decanting the liquid and resuspending with what is left in the tube) into antibiotic containing LB agar plates using a plastic spreader.
8. Incubate at 37°C overnight (approximately 16-18 hours).
9. Look for transformants the next morning.

*Source from OpenWetWare

Preparing overnight cultures

Preparing overnight cultures

During the procedure:
Work under flame at all times.

1. Prepare an overnight culture by inoculating one isolated single colony into 10mL of LB medium containing its respective antibiotic in a 50mL Falcon tube.
2. Incubate in a shaker overnight at 37°C.

Bacterial glycerol stock

Bacterial glycerol stock

Ingredients

• 50% sterile glycerol
• Cryogenic vials

1. Add 400µL of 50% glycerol to a cryogenic vial.
2. Add 600µL of culture sample to be stored.
3. Gently vortex the cryogenic vial to ensure the culture and glycerol is well-mixed. Alternatively, pipet to mix.
4. On the side of the vial list all relevant information, including: Part, vector, strain, date, researcher etc.
5. Store in a freezer box in a -80°C freezer.

Additional note:

• While it is possible to make a long term stock from cells in stationary phase, ideally your culture should be in logarithmic growth phase.



*Source from OpenWetWare

MINIPREP

MINIPREP

Ingredients:

• Buffer P1
• Buffer P2
• Buffer N3
• Buffer PB
• Buffer PE
• Milli-Q water

Before starting the procedure, check if:

• Ethanol has been added to the PE and P1 buffer
  • If added, there will be a tick on the lid.
  • If not, add according to instructions labelled on the bottle.

1. Spin down the overnight cultures at 10000 rpm at 4°C for 10 minutes using the communal centrifuge or 20mins at 4000rpm.
2. Discard the supernatant. Bacteria will be pelleted at the bottom of the 50mL Falcon tube.
3. Resuspend the pelleted bacteria with 250µL of Buffer P1 (always on ice).
4. Transfer the re-suspended bacteria to a fresh 2mL Eppendorf tube.
5. Add 250µL of Buffer P2 to the tube with bacteria and mix gently. Your sample should turn blue.
6. Incubate for 5 minutes at room temperature

(IMPORTANT: Do not allow more than 5 min incubation as this would degrade your plasmid)

7. Add 350µL of Buffer N3 and mix gently. Your sample should be colourless and should contain a white precipitant.
8. Centrifuge samples at 14000 rpm for 10 minutes using a tabletop centrifuge.
9. Transfer 750µL of the supernatant to a QIAprep Spin column (blue column).
10. Centrifuge at 11000 rpm for 1 minute using a tabletop centrifuge.
11. Pour the flow through again onto the column to increase the DNA concentration but do not close the cap tightly.
12. Spin it down quickly for 3 seconds using the table top centrifuge/let it sit for at least 10 minutes. Discard the flow through.
13. Add 500µL Buffer PB to the column.
14. Centrifuge at 13000 rpm for 30 seconds using a tabletop centrifuge.
15. Discard the flow through. Add 750µL Buffer PE to the column.
16. Incubate at room temperature for 5 minutes.
17. Centrifuge at 13000 rpm for 1 minute. Discard supernatant.
18. Centrifuge at 13000 rpm for 1 minute.
19. Transfer column to a fresh 1.5 mL Eppendorf tube.
20. Add 30µL of Milli-Q water (use lesser amount if concentration before miniprep is low)
21. Incubate for 5 minutes at room temperature.
22. Centrifuge at 11000 rpm for 1 minute.
23. Check concentration of DNA using Nanodrop.

*Source from Qiagen