Experiments

Objective - To join the restriction-digested pSB1C3 vector and BLIP insert into a single circular plasmid using T4 DNA ligase for downstream transformation and validation.

Materials & Equipment

- Restriction-digested pSB1C3 (vector)
- Restriction-digested BLIP fragment (insert)
- 10x T4 DNA ligase buffer
- ddH₂O (nuclease-free)
- T4 DNA ligase (keep on ice)
- 0.2 mL PCR/ligation tubes, tube rack
- Microcentrifuge (brief spin)
- Ice bucket / cold block
- Incubator or fridge set to 4 °C
- Competent cells for transformation (e.g., S17-1 or DH5α)
- Pipettes and sterile tips

Protocol

1. Label a sterile 0.2 mL tube with the sample ID and date.
2. Add 2.0 µL of digested pSB1C3 (vector).
3. Add 2.0 µL digested BLIP (insert).
4. Add 2.0 µL of 10x T4 DNA ligase buffer.
5. Add ddH₂O to bring the total volume to 19.5 µL.
6. Gently mix the mixture by pipetting up and down or flicking, then briefly spin it to collect liquid.
7. Add 0.5 µL of T4 DNA ligase (the enzyme was kept on ice prior to adding).
8. Briefly spin the tube and mix gently.
9. Incubate the reaction at 4 °C for 12-16 hours (overnight), and store the tube rack in the fridge during incubation.
10. After incubation, 10 µL of the ligation mix can be use for transformation. Store the remaining ~10 µL at -20 °C. [17]

Note: If ligation with 2 µL vector + 2 µL insert failed, the insert volume was increased while keeping the total volume ≤ 20 µL. Common insert: vector ratios included 3:1, 5:1, and 7:1.

Troubleshooting & Failures

Objective - To verify plasmid identity by digesting purified plasmid DNA with specific restriction enzymes and analyzing the resulting fragments via agarose gel electrophoresis to generate a simple restriction map.

Materials & Equipment

I. Restriction Digestion
- Purified plasmid DNA
- Restriction enzyme: EcoRI, PstI
- 10x CutSmart Buffer
- Sterile ddH2O
- Ice box

II. Agarose Gel Electrophoresis
- Agarose powder
- 1x TAE buffer
- DNA ladder
- 6x loading dye
- SYBR Safe (for staining)
- Gel casting tray and electrophoresis chamber
- UV transilluminator

Important Notes on Enzyme Use:
- Always add restriction enzymes last and keep them on ice at all times.
- Use a new pipette tip for each enzyme to avoid contamination.
- Since enzyme stocks contained 50% glycerol, use less than 1/10 of the total reaction volume to prevent inhibition.

Protocol

I. Restriction Digestion

1. Add 5 µL of plasmid DNA (~500 ng)to a labeled microcentrifuge tube.
2. Add 2 µL of 10x CutSmart Buffer.
3. Add sterile water to bring the total volume to 20 µL. Gently mix the mixture was tapping or pipetting and briefly spin down.
4. Add 0.5 µL (~5 units) of each restriction enzyme. Gently mix the mixture and spin down briefly again.
5. Incubate the reaction at 37 °C for 1 hour, then heat-inactivate at 80 °C for 20 minutes.
6. Store the reaction product at -20 °C until use.
7. Perform gel electrophoresis to confirm the digestion products.
Note: According to NEB, 1 unit of enzyme was defined as the amount required to digest 1 µg of λ DNA in a 50 µL reaction at 37 °C for 1 hour.

II. Agarose Gel Electrophoresis

1. Weigh the 0.5 g of agarose powder into a beaker.
2. Add 50 mL of 1x TAE buffer and mix gently.
3. Heat the solution on a hot plate until the agarose dissolves completely (the solution becomes clear).
4. Allow the solution to cool slightly (~60 °C).
5. Add 5 µL of SYBR Safe dye.
6. Pour the solution into a casting tray with a comb, and remove any bubbles.
7. Leave the gel to solidify at room temperature (~20-30 minutes).
8. Carefully removed the comb, leaving the gel ready for electrophoresis.
9. During sample preparation, add loading dye to the DNA samples with a dye ratio of 5:1.
10. Run the gel at 100 V for approximately 30 minutes, or until the bromophenol blue dye reaches the second-to-last line (around 600 bp in 1% agarose).
11. Visualize the DNA bands using a UV transilluminator. Capture the images, and attach the printout to the lab notebook.[17]

Troubleshooting & Failures

Objective - To amplify the BLIP-I and BLIP-II genes to verify successful transformation. This procedure generates sufficient DNA for downstream verification and analysis.

Materials & Equipment

- PCR machine
- VR primer
- VF2 primer
- Resuspended BLIP template DNA
- ddH₂O
- 2x Taq Mastermix

PCR cycling condition settings:

- Initial denaturation: 95 °C for 5 minutes.
- 35 cycles of:
→ Denaturation: 95 °C for 30 seconds.
→ Annealing: 55 °C for 30 seconds.
→ Extension: 72 °C for (1 minute per kb + 30 sec)—-2 minutes for BLIP-I and 2 minutes 30 sec for BLIP-II.
- Final extension: 72 °C for 10 minutes.
- Hold: 4 °C
- BLIP-I & BLIP-II concentration: 10 ng per reaction (typical range: 10-50 ng).

Protocol

1. Add 0.5 µL of VF2 primer and 0.5 µL of VR primer to a sterile PCR tube.
2. Add 1 µL of BLIP template DNA.
3. Add 3 µL of ddH₂O.
4. Add 5 µL of 2x Taq Mastermix (Add reagents from least to most concentrated).
5. Store the mixture temporarily at 4 °C. [17]

Notes: Remember to pipette mix the solution and mix the restriction enzymes before use.

Troubleshooting & Failures

Objective - To isolate pure fragments of BLIP-I, BLIP-II, and pSB1C3 using agarose gel electrophoresis.

Materials & Equipment

- DNA electrophoresis materials
- Gel-purified plasmids or ligation products
- Spin column
- Heat block
- Buffer B (dissolves gel)
- Buffer W1 & W2 (ethanol wash)
- ddH₂O (pH 7.0)

Protocol

Agarose Gel Electrophoresis

1. Weigh the 0.5 g of agarose powder into a beaker.
2. Add 50 mL of 1x TAE buffer and mix gently.
3. Heat the solution on a hot plate until the agarose dissolved completely (the solution becomes clear).
4. Allow the solution to cool slightly (~60 °C).
5. Add 5 µL of SYBR Safe dye.
6. Pour the solution into a casting tray with a comb, and remove any bubbles.
7. Leave the gel to solidify at room temperature (~20-30 minutes).
8. Carefully remove the comb, leaving the gel ready for electrophoresis.
9. During sample preparation, add loading dye to the DNA samples with a dye ratio of 5:1.
10. Run the gel at 100 V for approximately 30 minutes, or until the bromophenol blue dye reaches the second-to-last line (around 600 bp in 1% agarose).
11. Visualize the DNA bands using a UV transilluminator. Capture the images, and attach the printout to the lab notebook.
12. Excise desired bands using a clean (DNA-free) scalpel.

Gel Extraction

13. Add 500 µL of Buffer B to the gel slice, and incubate the mixture at 65 °C until the gel fully dissolves (~10 minutes, swirling every 2 minutes). Cool the solution to room temperature.
14. Transfer the dissolved gel solution to a spin column and centrifuge at 14,000 rpm for 1 minute. Discard flow-through.
15. Add 400 µL of Buffer W1, centrifuge for 1 minute. Discard flow-through.
16. Add 600 µL of Buffer W2 and spin for 1 minute. Discard flow-through.
17. Spin again at 14,000 rpm for 2 minutes, then open and air-dry for 2 minutes to remove residual ethanol.
18. Place the column into a new labeled tube. Add 25 µL of ddH₂O directly onto the membrane. Incubate the tube for 2 minutes at room temperature, then spin at 14,000 rpm for 2 minutes. Collect the supernatant, reapply to the membrane, and spin again for a second elution.
19. Measure DNA concentration (OD_260/280), and store the purified DNA at -20 °C.[17]

Troubleshooting & Failures

Objective - To isolate pure fragments of BLIP-I, BLIP-II, and pSB1C3 while minimizing DNA loss.

Materials & Equipment

- DNA electrophoresis materials
- Gel-purified plasmids or ligation products
- Spin column
- Heat block
- Buffer B (dissolves gel)
- Buffer W1 & W2 (ethanol wash)
- BE buffer (pH 7.0)

Protocol

Agarose Gel Electrophoresis

1. Weigh 0.5 g of agarose powder into a beaker.
2. Add 50 mL of 1x TAE buffer and mix gently.
3. Heat the mixture on a hot plate until the agarose dissolves completely (the solution becomes clear).
4. Allow the solution to cool slightly (~60 °C).
5. Add 5 µL of SYBR dye.
6. Pour the solution into a casting tray with a comb, and remove the bubbles if present.
7. Leave the gel to solidify at room temperature (~20-30 minutes).
8. Remove the comb carefully, and the gel is ready for electrophoresis.
9. During sample preparation, add loading dye to the DNA samples at a dye-to-sample ratio of 5:1.
10. Run the gel at 100 V for ~30 minutes, or until the bromophenol blue dye reaches the second-to-last line (~600 bp in 1% agarose).
11. Visualize DNA bands using a UV transilluminator, and capture and attach the images to the lab notebook.
12. Excise the desired DNA fragments from the agarose gel.
13. Transfer each gel slice (up to 300 mg) to a 1.5 mL microcentrifuge tube.
14. Add 500 µL of Buffer B to the sample, mix by vortexing, and incubate at 60 °C for 10 minutes (or until the gel slice is completely dissolved).
15. During incubation, the tube was mixed by vortexing every 2-3 minutes. The dissolved sample mixture was cooled to room temperature afterward.
16. Place a PG column into a collection tube. Apply the supernatant from step 15 to the PG column by decanting or pipetting.
17. Centrifuge the sample at 14,000 x g for 30 seconds.
18. Discard the flow-through, and place the PG column back into the same collection tube. (If the sample exceeded 800 µL, repeat the DNA binding step.)
19. Add 400 µL of Buffer W1 to the PG column and centrifuge at 14,000 x g for 30 seconds. Discard the flow-through, and place the column back into the same tube.
20. Add 600 µL of Buffer W2 (with ethanol added) to the PG column and centrifuge at 14,000 x g for 30 seconds. Discard the flow-through, and return the column to the same tube.
21. Centrifuge the column again at 14,000 x g for 2 minutes to remove residual Buffer W2.
22. Elute the DNA by placing the PG column into a clean 1.5 mL microcentrifuge tube.
23. Add 25 µL of Buffer BE (pH between 7.0 and 8.5) directly to the center of each PG column. Leave the sample to stand for 2 minutes, then centrifuge at 14,000 x g for 2 minutes. Repeat this elution step once. [6]

Troubleshooting & Failures

Objective - To prepare sterile LB agar plates and LB broth as general media for bacterial growth. These plates are used in future bacterial isolation and culture.

Materials & Equipment

- LB agar powder
- LB broth powder
- Distilled water (ddH₂O)
- Aluminum foil
- Autoclave tape
- Autoclave
- Stirring equipment (magnetic stirrer or heat and stir bar)
- Petri dishes
- Sterile centrifuge tubes or culture tubes
- Sterile bottles or flasks

Protocol

I. Prepared LB Agar (solid medium)

1. Mix 8 g LB agar powder with 250 mL ddH₂O in a suitable container (32 g LB Agar Powder: 1L ddH₂O).
2. Heat and stir until the powder is completely dissolved.
3. Cover the bottle opening with aluminum foil and secure it with autoclave tape (do not fully tighten the cap).
4. Sterilize by autoclaving at 121 °C for 30 minutes using moist heat.
5. Allow the LB agar to cool to approximately 65 °C, warm to the touch but not hot.
6. Pour the cooled agar into sterile Petri dishes, taking care not to let it solidify.

II. Prepared LB Broth (liquid medium)

1. Mix 6.25 g LB broth powder with 250 mL ddH2O (25 g LB Broth Powder:1 L ddH₂O).
2. Heat and stir until completely dissolved.
3. Cover the bottle opening with aluminum foil and secure it with autoclave tape (do not fully tighten the cap).
4. Sterilize by autoclaving at 121 °C for 30 minutes using moist heat.
5. Aliquot the sterile LB broth into sterile centrifuge tubes or culture tubes (e.g., 10 mL or 5 mL portions).
6. Store at 4 °C until use (do not heat after sterilization). [17]

Objective - To prepare selective media containing antibiotics for screening and maintaining bacteria carrying resistance genes. This way, we are able to verify if conjugation is successful.

Materials & Equipment

- LB agar or LB broth (prepared as in “Preparation of LB Agar and LB Broth,” Steps 1-4)
- Appropriate antibiotic stock solution
- Sterile Petri dishes or culture tubes
- Pipettes and sterile tips
- Stirring equipment (for agar)

Protocol

1. Prepare LB agar or broth following the protocol in “Preparation of LB Agar and LB Broth” (Steps 1-4).
2. Allow the medium to cool to a temperature suitable for adding antibiotics (~45-55 °C).
3. Add the antibiotic at a 1:1000 dilution (Antibiotic: LB = 1:1000). Mix gently to avoid bubbles.
4. Pour agar into sterile Petri dishes or aliquot broth into sterile tubes.
5. Allow the agar to solidify or store the broth at 4 °C until use.

Working Concentration for Each Antibiotic [17]

Objective - To obtain well-isolated single bacterial colonies from a mixed culture. This ensures the establishment of clonal populations that can be used reliably in downstream experiments and characterization.

Materials & Equipment

- LB agar plates
- Bacterial suspension (from glycerol stock, broth culture, etc.)
- Bunsen burner (or alcohol lamp)
- Parafilm
- Permanent marker (for labeling)
- Incubator (set to 37 °C)
- (Quadrant Streak Method) Inoculating loop (reusable) or sterile disposable toothpicks
- (Spreading Method) Glass spreader (hockey stick)
- (Spreading Method) 95% ethanol (for spreader sterilization)
- (Spreading Method) Micropipette and sterile tips (for adding suspension, e.g., 100 µL)

Protocol

I. Quadrant Streak Method

1. Label the bottom of an LB agar plate.
2. Sterilize an inoculating loop by flaming it over a Bunsen burner until red hot and allow it to cool before use, or use a sterile disposable toothpick.
3. Dip into the bacterial suspension and streak a small section of the plate (first quadrant).
4. Sterilize the loop again, streak from the first quadrant into the second quadrant, spreading fewer cells.
5. Repeat for the third and fourth quadrants to obtain isolated colonies.
6. Incubate at 37 °C for 12-16 hours.
7. Pick well-isolated colonies for further experiments or seal the plate with parafilm and store it at 4 °C for further use.

II. Spreading Method

1. Label the bottom of the agar plate with bacterial host, treatment, antibiotic resistance, your name, and date.
2. Sterilize the glass spreader (hockey stick) as follows:
→ Immerse the spreader in 95% ethanol.
→ Remove it and allow excess ethanol to drip off.
→ Pass the spreader briefly through a flame to sterilize.
→ Allow it to cool before using it to spread the bacterial suspension.
3. Drop a suitable volume of bacterial suspension onto the plate (commonly 100 µL).
4. Use the cooled spreader to evenly spread the suspension across the agar surface until no visible liquid remains.
5. Allow the bacterial suspension to dry briefly on the plate.
6. Incubate the plate at an appropriate temperature (usually 37 °C) for 12-16 hours.
7. Pick well-isolated colonies for further experiments or seal the plate with parafilm and store it at 4 °C for further use.

Troubleshooting & Failures

I. Quadrant Streak Method

II. Spreading Method

Objective - To revive lyophilized E. coli S17-1 cells and establish an active bacterial culture. Since the cells are freeze-dried for shipping, proper rehydration and resuspension are required prior to use.

Materials & Equipment

- Sterile water
- Sterile broth (0.5 mL - 10 mL)
- LB agar plates (2)
- Bacterial sample
- Heat source (e.g., Bunsen burner or heating block)
- Sterile forceps/tweezers
- Micropipette and sterile pipette tips
- Sterile L-shaped spreader (hockey stick spreader)
- Shaker incubator (37 °C)
- Standard incubator (37 °C)

Protocol

1. Heat the outer tube at the tip (do not expose the bacterial pellet to heat).
2. Place a drop of sterile water on the heated area (thermal expansion helps loosen the outer tube).
3. Use sterile forceps to crack the outer tube tip carefully.
4. Gently remove the inner tube.
5. Remove the cotton plug.
6. Add 0.3-0.5 mL sterile broth into the inner tube to resuspend the bacterial pellet into a suspension.
7. Prepare 1 tube of 5-10 mL LB broth and 2 LB agar plates.
8. Pipette 0.1 mL of the bacterial suspension onto each LB agar plate.
9. Spread evenly using a sterile L-shaped spreader (hockey stick spreader).
10. Transfer the remaining suspension into the 5-10 mL LB broth tube.
11. Incubate:
- LB agar plates: 16 hours at 37 °C.
- LB broth: 16 hours at 37 °C with shaking at 200 rpm. [27]

Troubleshooting & Failures

Objective - To prepare for long-term storage of E. coli at -80 °C using glycerol as a cryoprotectant. Storing bacteria in glycerol stock preserves it for potentially years, preventing cell death and denaturing.

Materials & Equipment

- 1.5 mL microcentrifuge tube or screwcap tube
- 100% glycerol (to be diluted with ddH₂O to 50%)
- E. coli S17-1 culture grown in broth
- Micropipettes and sterile tips
- Dry ice (for snap freezing, optional)
- -80 °C freezer

Protocol

1. Prepare 50% glycerol by diluting 100% glycerol with ddH₂O.
2. In a sterile microcentrifuge tube/screwcap tube, add 500 µL of 50% glycerol and 500 µL of bacterial culture.
3. Mix gently by pipetting up and down (avoid bubbles).
4. (Optional) Snap-freeze the vial using dry ice.
5. Store the glycerol stock at -80 °C. [1]

Troubleshooting & Failures

Objective -To recover viable bacterial cells from -80 °C glycerol stocks and establish active cultures for experimental use. This step provides a consistent starting material and ensures strain integrity for downstream applications.

Materials & Equipment

- Glycerol stock (from -80 °C freezer)
- LB agar plates
- LB broth
- Sterile culture tubes
- Pipettes and sterile tips
- Sterile toothpicks or inoculating loops
- Shaking incubator (37 °C, 250 rpm)
- Standard incubator (37 °C)

Protocol

1. Retrieve glycerol stock from -80 °C and keep it on ice.
2. Use a sterile loop, pipette tip, or toothpick to take a small sample from the stock.
3. Perform quadrant streaking on an LB agar plate.
4. Incubate the plate overnight at 37 °C.
5. The next day, check for isolated single colonies.
6. Fill a sterile culture tube with LB broth.
7. Use a sterile pipette tip, toothpick, or loop to pick the chosen colony.
8. Transfer the colony into the LB broth.
9. Incubate the culture overnight at 37 °C with shaking at 250 rpm. [28]

Troubleshooting & Failures

Objective - To prepare E. coli S17-1 cells for transformation by making them chemically competent with CaCl2 treatment. This step increases cell membrane permeability and facilitates efficient uptake of plasmid DNA in subsequent experiments.

Materials & Equipment

- Overnight culture of E. coli S17-1
- LB broth
- Pre-chilled 1.5 mL microcentrifuge tubes
- 10% CaCl₂ solution (ice cold)
- 15% glycerol + 1.11 g CaCl₂ solution (for glycerol stock)
- Spectrophotometer (OD₆₀₀ measurement)
- Shaking incubator (37 °C, 200 rpm)
- Refrigerated centrifuge
- Ice bucket

Protocol

1. Inoculate 0.6 mL of the overnight culture into 12 mL LB broth.
2. Incubate at 37 °C with shaking (200 rpm) for 4 hours.
3. Measure OD₆₀₀ and harvest when the culture reaches ~0.4-0.6.
4. Place the culture on ice for 10 minutes.
5. Transfer 1 mL aliquots into pre-chilled 1.5 mL microcentrifuge tubes.
6. Centrifuge at 5000 x g for 10 minutes at 4 °C. Discard the supernatant.
7. Resuspend the pellet in 1 mL ice-cold 10% CaCl₂. Incubate on ice for 1 hour.
8. Centrifuge at 4000 x g for 15 minutes at 4 °C. Discard the supernatant.
9. Resuspend the pellet in 100 µL ice-cold 10% CaCl₂ for immediate heat-shock transformation.
10. Alternatively, resuspend in 100 µL of 15% glycerol + CaCl₂ solution for long-term storage at -80 °C. [29][30]

Objective -To introduce foreign DNA (plasmid) into competent E. coli cells by temporarily making their cell membranes permeable, allowing the DNA to enter and produce transformed bacteria.

Materials & Equipment

- Competent E. coli cells (pre-chilled)
- Resuspended DNA (plasmid or insert)
- 1.5 mL microcentrifuge tubes (pre-chilled)
- Ice and ice bucket
- Pipettes and sterile tips
- Water bath or heat block (42 °C)
- SOC or LB media (pre-warmed to room temperature)
- Sterile Petri plates with appropriate agar and antibiotic
- Sterile glass spreaders or glass beads
- Shaking incubator (37 °C, 200-300 rpm)
- Centrifuge capable of 6800 x g
- Sterile loop or toothpick for picking colonies
- Colony PCR reagents and equipment (if verifying transformants)
- Glycerol for making stocks (optional)

Protocol

1. Thaw competent cells on ice.
2. Pipette 50 µL of competent cells into a 1.5 mL tube (pre-chilled).
3. Pipette 1 µL of resuspended DNA into a 1.5 mL tube: pipette from well into appropriately labeled tube. Gently pipette up and down a few times. Keep all tubes on ice.
4. Close 1.5 mL tubes, incubate on ice for 20-30 minutes: tubes may be gently agitated/flicked to mix solution, but return to ice immediately.
5. Heat-shock tubes at 42 °C for 30-45 seconds.
6. Incubate on ice for 5 minutes.
7. Pipette 950 µL LB media into each transformation: LB media is stored at 4 °C, but can be warmed to room temperature before use. Check for contamination.
8. Incubate at 37 °C for 1 hour, shaking at 200-300 rpm.
9. Take the plate out of the fridge to warm to room temperature.
10. Pipette 100 µL of each transformation onto LB plates. Spread with a sterilized spreader or glass beads immediately.
11. Spin down cells at 6800 x g for 3 minutes.
12. Discard 800 µL of the supernatant. Resuspend the cells in the remaining 100 µL, and pipette each transformation onto Petri plates. Spread with a sterilized spreader or glass beads immediately. This increases the chance of getting colonies from lower-concentration DNA samples.
13. Incubate transformations overnight (14-18 hr) at 37 °C: incubate the plates upside down (agar side up). If incubated for too long, colonies may overgrow, and the antibiotics may start to break down: untransformed cells begin to grow.
14. Pick single colonies: pick single colonies from the transformation. Perform colony PCR to verify part size, make a glycerol stock, grow up cell cultures, and miniprep. [16]

Troubleshooting & Failures

Objective -To isolate and purify plasmid DNA from bacterial cells using alkaline lysis followed by silica resin column purification. The resulting pure, high-quality plasmid DNA can then be used for a variety of critical downstream applications.

Materials & Equipment

- Cultured bacterial solution
- Genedirex Plasmid miniPREP Kit (Cat No. NA005-0100):
→ Buffer S1 with RNase (final conc. 0.1 mg/mL)
→ Buffer S2
→ Buffer S3
→ Spin column
→ Buffer W1
→ Buffer W2 (with ethanol)
→ Sterile ddH₂O (pH 7.0-8.5) or Buffer BE (Tris-based elution buffer)

Protocol

1 Bacterial Cells Harvesting
1. Transfer the bacterial culture (up to 1.5 mL) to a microcentrifuge tube.
2. Centrifuge at 14,000 x g for 1 minute and discard the supernatant.
Step 2 Resuspension
1. Resuspend the pellet of bacterial cells in 200 μL of Buffer S1 (RNase A added).
Step 3 Lysis
1. Add 200 μL of Buffer S2 and mix thoroughly by inverting the tube 10 times (do not vortex). Stand at room temperature for 2 minutes or until the lysate is homogeneous.
Step 4 Neutralization
1. Add 300 μL of Buffer S3 and mix immediately and thoroughly by inverting the tube 10 times (do not vortex).
2. Centrifuge at 14,000 x g for 3 minutes.
Step 5 Binding
1. Place a PM column in a Collection Tube. Apply the supernatant (from step 4) to the PM column by decanting or pipetting.
2. Centrifuge at 14,000 x g for 30 seconds, discard the flow-through, and place the PM column back into the same Collection Tube.
Step 6 Wash
1. Add 400 μL of Buffer W1 into the PM column.
2. Centrifuge at 14,000 x g for 30 seconds.
3. Discard the flow-through and place the PM column back into the same Collection Tube.
4. Add 600 μL of Buffer W2 (ethanol added) into the PM column.
5. Centrifuge at 14,000 x g for 30 seconds.
6. Discard the flow-through and place the PM column back into the same Collection Tube.
7. Centrifuge at 14,000 x g again for 2 minutes to remove the residual Buffer W2.
Step 7 Elution
1. Place the PM column in a clean 1.5 mL microcentrifuge tube. Add 50-200 μL of Buffer BE or H₂O (pH 7.0-8.5) to the center of the PM column. Let it stand for 2 minutes and centrifuge at 14,000 x g for 2 minutes.
2. Take the flow-through, add it again to the center of the PM column, and centrifuge at 14,000 x g for 2 minutes. [4]

Objective - To resuspend dried DNA samples from the distribution kit into solutions for subsequent transformation into competent cells. By properly rehydrating the DNA (containing cresol red dye for visual confirmation), we ensure accurate handling and avoid cross-contamination. The resuspended DNA is then used for bacterial transformation, colony growth, minipreparation, and glycerol stock creation, enabling reliable downstream applications such as restriction digest and sequencing.

Materials & Equipment

- Distribution kit DNA plate (dried DNA samples with cresol red dye)
- ddH₂O (pH 7.0)
- Pipettes and sterile tips
- Sterile 1.5 mL microcentrifuge tubes

Protocol

Note: There is an estimated 2-3 ng of DNA in each well. When following this protocol, assume that you are transforming with 200-300 pg/µL.

1. With a pipette tip, punch a hole through the foil cover into the corresponding well of the part that is needed. Ensure the plate is properly oriented. Do not remove the foil cover, as doing so can lead to cross-contamination between the wells.
2. Pipette 10 µL of dH₂O (distilled water) into the well. Pipette mix a few times and let sit for at least 5 minutes to ensure the dried DNA is fully resuspended. The resuspension appears red, as the dried DNA contains cresol red dye. [14]

Objective - To prepare LB agar plates with X-Gal for blue/white colony screening of recombinant bacterial colonies.

Materials & Equipment

- LB agar
- Petri dish
- Bunsen Burner
- Thermo Scientific X-Gal
- Hockey Stick

Protocol

1. Pour sterile warm LB agar (about 25 mL) into a Petri dish.
2. Dry opened LB plates at room temperature around bunsen burner for about 30 minutes.
3. Add 40 μL of the Thermo Scientific X-Gal Solution (20 mg/mL), ready-to-use (Cat #R0941).
4. Spread evenly on the plate with a Hockey stick.
5. Dry open LB plates at room temperature around the Bunsen burner for about 30 minutes. [35]

Objective - To perform solid surface conjugation between E. coli S17-1 (carrying BLIP-I or BLIP-II) and E. coli DH5α (Ampicillin-resistant), allowing horizontal gene transfer through cell-to-cell contact on LB agar. This experiment evaluates the effectiveness of BLIP-I and BLIP-II in inhibiting β-lactamase activity after conjugation.

Materials & Equipment

Bacterial Strains

- Donor: E. coli S17-1 carrying BLIP-I or BLIP-II (Chloramphenicol-resistant)
- Recipient: E. coli DH5α (Ampicillin-resistant)

Selective agar plates:

- LB + Ampicillin+X-gal (50 µg/mL)
- LB + Chloramphenicol+X-gal (25 µg/mL)
- LB + Ampicillin + Chloramphenicol+X-gal
- PBS buffer (pH 7.4) – for washing and resuspension steps

DAP (optional) – added only if donor requires diaminopimelic acid for growth
Equipment and Consumables

- Microcentrifuge tubes (1.5 mL)
- Micropipettes and sterile tips
- Centrifuge (capable of ~6800 x g)
- Spectrophotometer (for OD₆₀₀ measurement)
- Sterile inoculating loops or pipette tips (for mixing donor and recipient on plate)
- Vortex mixer
- 37 °C incubator

Protocol

Step 1. Prepare overnight cultures:
1. Grow E. coli S17-1 (BLIP-I / BLIP-II) and E. coli DH5α (Ampicillin-resistant) separately in LB broth at 37 °C, shaking at 250 rpm overnight until visibly turbid.
Step 2. Remove residual antibiotics:
1. Centrifuge 1 mL of each overnight culture at ~6800 x g for 1 minute, then discard the supernatant.
Step 3. Wash cells with PBS:
1. Resuspend each pellet in 1 mL PBS, pipette up and down to mix, centrifuge again, and discard the supernatant. Repeat once to ensure all antibiotics are removed.
Step 4. Normalize cell densities:
1. Resuspend each pellet in 500 µL PBS and measure OD₆₀₀. Adjust cell densities to approximately equal levels for a balanced donor-recipient ratio.
Step 5. Mix donor and recipient:
1. In a sterile microcentrifuge tube, mix donor and recipient cells in a 1:1 ratio (total volume ≈ 100 µL). Mix gently by pipetting.
Step 6. Spot mixture onto the plate:
1. Spot 50–100 µL of the mixture onto an LB agar plate without antibiotics (with DAP if required). Do not spread; allow the spot to air-dry under sterile conditions.
Step 7. Incubate for conjugation:
1. Incubate the plate at 37 °C for 4–6 hours to allow cell-to-cell contact and plasmid transfer.
Step 8. Recover conjugation mixture:
1. After incubation, scrape the bacterial spot into 1 mL PBS and vortex briefly to resuspend.
Step 9. Plate on selective media:
1. Dilute the suspension as needed and plate onto LB + Ampicillin + Chloramphenicol selective agar to isolate transconjugants.
Step 10. Incubate and observe:
1. Incubate selective plates overnight at 37 °C. Colonies confirming successful conjugation can be observed the next day. [32][33][34]

Troubleshooting & Failures

1. Addgene. Addgene: Protocol - How to Create a Bacterial Glycerol Stock. https://www.addgene.org/protocols/create-glycerol-stock/
2. The Streak Plate Protocol. ASM. https://asm.org/asm/media/protocol-images/the-streak-plate-protocol.pdf (accessed 2025-10-03)
3. Bacteriology Culture Guide. www.atcc.org. https://www.atcc.org/resources/culture-guides/bacteriology-culture-guide
4. Plasmid miniPREP Kit. GeneDireX, Inc. https://www.genedirex.com/product/plasmid-miniprep-kit/
5. Bacteriology Culture Guide. www.atcc.org. https://www.atcc.org/resources/culture-guides/bacteriology-culture-guide
6. PCR Clean-Up & Gel Extraction Kit. Genedirex.com https://www.genedirex.com/wp-content/uploads/2021/01/NA006-0100.pdf (accessed 2025-10-02)
7. FUNDAMENTAL TECHNIQUES in CELL CULTURE LABORATORY HANDBOOK 3 RD EDITION. https://www.sigmaaldrich.com/deepweb/assets/sigmaaldrich/marketing/global/documents/425/663/fundamental-techniques-in-cell-culture.pdf
8. Why do I get smeared PCR products? Qiagen.com. https://www.qiagen.com/us/resources/faq/87
9. PCR Troubleshooting Guide | Thermo Fisher Scientific - US. Thermofisher.com. https://www.thermofisher.com/tw/zt/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/pcr-education/pcr-reagents-enzymes/pcr-troubleshooting.html (accessed 2025-10-03)
10. Sambrook, J. and Russell, D.W. (2001) Molecular Cloning A Laboratory Manual. 3rd Edition, Vol. 1, Cold Spring Harbor Laboratory Press, New York. - References - Scientific Research Publishing. Scirp.org. https://www.scirp.org/reference/ReferencesPapers.aspx?ReferenceID=1765722 (accessed 2025-10-01)
11. GPP Web Portal - Directory cannot be listed. Broadinstitute.org. https://portals.broadinstitute.org/gpp/public/dir/download (accessed 2025-10-03)
12. Bench Guide. Qiagen.com. https://www.qiagen.com/us/knowledge-and-support/knowledge-hub/bench-guide/ (accessed 2025-10-03)
13. Storing Bacterial Samples for Optimal Viability | Thermo Fisher Scientific - US. Thermofisher.com. https://www.thermofisher.com/us/en/home/industrial/microbiology/microbiology-learning-center/storing-bacterial-samples-for-optimal-viability.html
14. Distribution Kit DNA Resuspension. https://static.igem.org/mediawiki/2020/5/5b/T--UofUppsala--Distribution_kit_DNA_resuspension.pdf (accessed 2025-10-04)
15. Brown, T. A. Genomes. Nih.gov. https://www.ncbi.nlm.nih.gov/books/NBK21128/
16. Help: Protocols/Transformation - parts.igem.org. Igem.org. https://parts.igem.org/Help:Protocols/Transformation (accessed 2025-10-05)
17. 2025 iGEM Laboratory Guidelines. NYCU.
18. Troubleshooting: Bacterial Transformation | GoldBio. Goldbio.com. https://goldbio.com/articles/article/troubleshooting-bacterial-transformation?srsltid=AfmBOopy9bMNubGMQMsLgd4QAc5RqziSg8M5K9V5Vg3qTsuiHXcy7fiq& (accessed 2025-10-05)
19. Troubleshooting: Satellite Colonies. Goldbio.com. https://goldbio.com/articles/article/satellite-colonies?srsltid=AfmBOoqTn3_LS-M1z1MN6k6KufTGYsrnfJYLi6hYWCsJrRpLWToMHvhn& (accessed 2025-10-05)
20. Bacterial Transformation Workflow | Thermo Fisher Scientific - US. Thermofisher.com. https://www.thermofisher.com/tw/zt/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/transformation/bacterial-transformation-workflow (accessed 2025-10-05)
21. Troubleshooting Guide. Edvotek.com. https://www.edvotek.com/transformation-troubleshooting-guide?srsltid=AfmBOopbC9MGzTWFGoqM00zpx427wpjxp
22. Addgene: Protocol - Bacterial Transformation. Addgene.org. https://www.addgene.org/protocols/bacterial-transformation
23. Yang, Y.; Liu, M.; Wang, T.; Wang, Q.; Liu, H.; Xun, L.; Xia, Y. An Optimized Transformation Protocol for Escherichia Coli BW3KD with Supreme DNA Assembly Efficiency. Microbiology Spectrum 2022, 10 (6). https://doi.org/10.1128/spectrum.02497-22
24. New England Biolabs. Neb.com. https://www.neb.com/en/tools-and-resources/troubleshooting-guides/troubleshooting-transformation-reactions?srsltid=AfmBOoryN8Q9yFUb-phw_cOrlIIcYTjk5vnN41GgCcs52f2HV6LC_3Aj (accessed 2025-10-05)
25. E. Coli Competent Cells. https://worldwide.promega.com/-/media/files/resources/protocols/technical-bulletins/0/e-coli-competent-cells-protocol.pdf
26. Bacterial Transformation Troubleshooting Guide | Thermo Fisher Scientific - US. Thermofisher.com. https://www.thermofisher.com/tw/zt/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/transformation/bacterial-transformation-troubleshooting-guide.html (accessed 2025-10-05)
27. 冷凍乾燥管之臨時存放及開封活化說明 (Temporary Storage and Revival of Lyophilized Vials). Food Industry Research and Development Institute. PDF Link
28. Reviving Glycerol Stock. Technische Universiteit Eindhoven University of Technology. https://static.igem.org/mediawiki/2015/a/a1/TU_Eindhoven_Protocols_Streaking_Glycerol_Stock.pdf
29. How to Make Chemically Competent E. coli. Youtu.be. https://youtu.be/H4X-5Bg0bE8?si=hEPtQC9LKfNHRjSu (accessed 2025-09-13)
30. How to Make Competent Cells: Protocols and Tips. Zymo Research International. https://zymoresearch.eu/blogs/blog/how-to-make-competent-cells-protocols-and-tips?srsltid=AfmBOorWrTUbyt60IlevDWfroiJWyt5QnEfbkKV-RtjWFN9gNSiDxc4Z (accessed 2025-09-13)
31. No Bands - Genotyping. The Jackson Laboratory. https://www.jax.org/jax-mice-and-services/customer-support/technical-support/genotyping/troubleshooting/no-bands
32. Barrick Lab: Protocols Conjugation. barricklab.org. https://barricklab.org/twiki/bin/view/Lab/ProtocolsConjugation
33. Introductory Bacterial Conjugation TEACHER'S MANUAL and STUDENT GUIDE Strain I Streptomycin-Resistance Gene on Chromosome Strain II Ampicillin-Resistance Gene on Plasmid. PDF Link
34. Elston, K. M.; Phillips, L. E.; Leonard, S. P.; Young, E.; Holley, J. C.; Tasneem Ahsanullah; McReynolds, B.; Moran, N. A.; Barrick, J. E. The Pathfinder Plasmid Toolkit for Genetically Engineering Newly Isolated Bacteria Enables the Study of Drosophila-Colonizing Orbaceae. ISME Communications 2023, 3 (1). https://doi.org/10.1038/s43705-023-00255-3
35. Preparation of X-Gal/IPTG LB Agar Plates for Blue/White Colony Screening. https://documents.thermofisher.com/TFS-Assets/BID/Methods-&-Protocols/75-preparation-of-x-gal-iptg-lb-agar-plates-for-blue-white-colony-screening.pdf(accessed 2025-10-08).