Notebook

Our Method

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1. Prepare Inoue transformation buffer (chilled to 0°C)
2. Choose a single bacterial colony 2-3 mm in diameter from a plate that has been incubated for 16-20 hours at 37°C. Inside a 250-mL flask, transfer the colony into 25 mL of SOB or LB medium. Incubate for 6-8 hours at 37°C with shaking at 250-300 rpm.
3. At around 6pm, fill three 1-liter flasks with 250 mL of SOB, and inoculate from the starter culture. The first flask will receive 10 mL of starter culture, the second flask will receive 4 mL, and the third flask will receive 2 mL. Incubate all three flasks overnight at 18-22°C with moderate shaking (150-250 rpm).
4. The next morning, read the OD₆₀₀ from all three flasks, and monitor every 45 minutes
5. Put the culture vessel in an ice water bath for 10 minutes when the OD₆₀₀ of one of the cultures reaches 0.55
6. Centrifuge the cells at 2500g for 10 minutes at 4°C to harvest them.
7. Decant the supernatant and keep the open centrifuge bottle on a stack of paper towels for 2 minutes, and use a vacuum aspirator to remove any remaining drops of medium from the walls of the centrifuge bottle.
8. Gently resuspend the cells in 80 mL of ice-cold Inoue transformation buffer
9. Centrifuge the cells again at 2500g for 10 minutes at 4°C to harvest them.
10. Again decant the supernatant, let cells rest on a stack of paper towels for 2 minutes, and aspirate any remaining drops of medium.
11. Gently resuspend the cells in 20mL of ice-cold Inoue transformation buffer
12. Add in 1.5 mL of DMSO, swirl the suspension to mix and store on ice for 10 minutes.
13. Working quickly, dispense aliquots of the suspensions into chilled, sterile microcentrifuge tubes. Immediately snap-freeze the cells by immersing the tightly closed tubes in a liquid nitrogen bath. Store the tubes in the freezer at -70°C until needed.
14. When needed, remove a tube of competent cells from the freezer. Thaw by holding in the palm of the hand. Just as the cells thaw, transfer the tube to an ice bath. Store on ice for 10 minutes.
15. Using a chilled, sterile pipette tip, transfer the cells to chilled, sterile 17 x 100-mm polypropylene tubes. Store the competent cells on ice. Include all positive and negative controls
16. Add up to 25 ng of transforming DNA per 50 µl competent cells. Gently swirl the tubes several times to mix. Create at least two control tubes for each transformation experiment, including a tube of competent cells that receives a known amount of a standard preparation of superhelical plasmid DNA and a tube receiving no plasmid at all. Keep the tubes on ice for 30 minutes.
17. Move the tubes to a rack placed in a preheated 42°C circulating water bath. Keep the tubes in the rack for exactly 90 seconds. Do not shake the tubes.
18. Quickly move the tubes to an ice bath and allow the competent cells to cool for 1-2 minutes.
19. To each tube, add 800 µl of SOC medium and warm the cultures to 37°C in a water bath, then transfer the tubes to a 37°C shaking incubator. Incubate the cultures for 45 minutes, allowing the bacteria to recover and express the antibiotic resistance marker encoded by the plasmid.
20. Transfer the appropriate volume (up to 200 µl per 90-mm plate) of transformed competent cells onto agar SOB medium that contains 20mM MgSO₄ and the appropriate antibiotic.
21. Keep the plates at room temperature until the liquid is absorbed.
22. Invert the plates and keep them incubated at 37°C. Transformed colonies should appear in 12-16 hours.

Materials

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• DMSO
• Inoue transformation buffer (chilled to 0°C)
• Plasmid DNA
• SOB agar plates containing 20mM MgSO4 and appropriate antibiotic
• SOB medium for initial growth of culture
• SOB medium for growth of culture to be transformed
• SOC medium

Making and Pouring a Gel for Gel Electrophoresis

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1. Measure out agarose for a 1% agarose gel. For a 35 ml gel tray, use 0.35g of agarose.
2. Use graduated cylinder to measure 35ml of TBE buffer.
3. Pour both into a small microwaveable erlenmeyer flask, swirl.
4. Using heat protecting gloves, heat mixture in microwave in 15 second increments with a small beaker as a lid until the mixture boils.
5. Check mixture for clarity, agarose should be completely dissolved. If not, keep microwaving until dissolved.
6. Add 3.5ml of gel red.
7. Make sure the comb is placed properly in the gel tray.
8. Using a paper towel to hold the flask, gently pour gel until just under the top of the comb slots.
9. Wait about 40 minutes, until the gel is completely solid and cooled.

Loading and Running a Gel for Electrophoresis

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1. Once solidified, place the agarose gel in the gel box (electrophoresis unit)
2. Fill gel box with TAE buffer until gel is completely covered
3. Carefully load first well with 5 μl of molecular weight ladder
4. Pipette 5 μl sample onto parafilm stretched over a tray
5. Pipette 1 μl loading dye onto sample, slowly pull mixture up and down with the micropipette
6. Pull 5 μl of sample up from the mixture, and carefully pipette sample into next empty well
7. Write down your sample ID with well number
8. Repeat steps 4-7 with all remaining samples
9. Run the gel at 80 V until the dye line is approximately 80% of the way down the gel. A typical run time is around 1-1.5 hours.
10. Turn off power, disconnect the electrodes from the power supply, and then carefully remove the gel from the gel box

Procedure

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1. Centrifugation:
   • Centrifuge 1.5 ml of overnight E. coli culture at >13,000 rpm for 1 minute at room temperature (15-25°C)
   • Ensure the culture's OD is between 2.0 and 4.0.
2. Medium Removal:
   • Carefully decant or pipette off the supernatant.
   • Invert the tubes on a paper towel to remove any remaining medium.
3. Add Lysis Solution:
   • Add 400 µl of ice-cold Complete Lysis Solution to the pelleted bacterial cells.
   • Ensure the solution remains cold (<4°C) to optimize DNA yield.
4. Mixing:
   • Vortex the tube on the highest setting for 30 seconds.
   • Ensure no cell clumps remain.
5. Incubation:
   • Incubate the lysate at room temperature (15-25°C) for 3 minutes.
   • The lysate should appear nonviscous and slightly cloudy.
6. Transfer Lysate:
   • Transfer the lysate to a QuickLyse spin column by decanting or pipetting.
7. Initial Centrifugation:
   • Centrifuge the spin column for 30-60 seconds at 13,000 rpm.
   • There's no need to decant the flow-through.
8. Wash the Column:
   • Add 400 µl of diluted Buffer QLW to the QuickLyse spin column.
   • Centrifuge for 30-60 seconds at 13,000 rpm and discard the flow-through.
9. Dry the Column:
   • Place the spin column back in the waste tube.
   • Centrifuge for 1 minute at 13,000 rpm to dry the column.
10. Elution of DNA:
    • Transfer the spin column into a clean collection tube.
    • Add 50 µl of Buffer QLE to the center of the column.
    • Centrifuge for 30-60 seconds at 13,000 rpm.
11. Storage:
    • Use the eluted DNA immediately or store it at -20°C for future use.

Materials

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• QuickLyse Lysis Tubes (2 ml)
• Complete Lysis Solution (ice cold)
• Diluted Buffer QLW
• Overnight E. coli culture (1.5 ml, OD 2.0-4.0)
• Centrifuge (>13,000 rpm)
• Vortex mixer
• Pipettes and sterile tips
• Buffer QLE (for elution)

Procedure

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1. LB-Agar Media Preparation:
   • Measure 37 g of pre-mixed LB-agar powder per 1 L of media.
   • Adjust the quantity based on the number of plates you plan to make. Example: For 220 mL of media (enough for 20 plates), measure 8.14 g of LB-agar powder.
2. Mixing the Agar Solution:
   • Transfer the measured LB-agar powder into a sterilizable bottle.
   • Add sterile water to the bottle (e.g., 220 mL for 220 mL of media).
   • Swirl the bottle to mix until you achieve a colloid.
3. Autoclaving:
   • Loosely cover the bottle with the cap or aluminum foil (do not create an air-tight seal).
   • Label the bottle with tape (include initials, date, and contents).
   • Autoclave the mixture at 121°C and 20 psi for 30 minutes.
4. Plate Preparation Station:
   • While autoclaving, prepare a clean lab bench area with a flame source for sterile plate pouring.
   • Spray down the bench with 70% ethanol and wipe with paper towels.
   • Stack and label the sterile plates with the date, media type, and antibiotic.
5. Cooling the Agar:
   • After autoclaving, allow the agar to cool in the autoclave for ~10 minutes with the door cracked open.
   • Transfer the bottle to a 60°C water bath. Submerge ~75% of the bottle and leave for at least 5 minutes.
6. Antibiotic Addition:
   • Prepare the antibiotic stock (e.g., 100 mg of ampicillin in 1 mL sterile water for 100 mg/mL concentration).
   • Add the appropriate amount of antibiotic to the cooled (~60°C) molten agar. Example: For 220 mL of LB-agar, add 220 µL of 100 mg/mL Ampicillin to achieve a 100 µg/mL working concentration.
   • Swirl the agar bottle gently to ensure even distribution of the antibiotic.
7. Pouring Plates:
   • Flame your working area.
   • Open one plate at a time near the flame and pour the molten agar. For the first plate, measure the amount with a pipette to get an estimate of the volume needed (typically 5-10 mL per plate).
   • For the remaining plates, pour directly from the bottle.
   • Swirl the plates to remove bubbles and ensure even distribution. Cap each plate after pouring.
8. Solidification and Drying:
   • Leave the plates on the bench to solidify (takes ~30 minutes at room temperature).
   • Allow the plates to dry overnight at room temperature to prevent condensation. Place the plates in a plastic bag with absorbent material and store them at 4°C until use.
9. Antibiotic Testing:
   • After the plates have dried, test their antibiotic function by streaking two plates:
     • Plate 1: Streak a strain known to be resistant to the antibiotic.
     • Plate 2: Streak a strain not resistant to the antibiotic.
   • Incubate overnight at the appropriate temperature and check for growth.

Materials

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• Pre-mixed LB-agar powder (37 g/L):
  • 5 g Yeast extract
  • 10 g Peptone from casein
  • 10 g Sodium chloride (NaCl)
  • 12 g Agar-agar
• Sterile H₂O (1 L)
• Sterile plates (e.g., 60 mm x 15 mm or 100 mm x 15 mm)
• Antibiotic stock (1000x concentration)
• Optional Antibiotics & Concentrations:
  • Ampicillin (100 mg/mL, working concentration 100 µg/mL)
  • Carbenicillin (100 mg/mL, working concentration 100 µg/mL)
  • Chloramphenicol (25 mg/mL in EtOH, working concentration 25 µg/mL)
  • Kanamycin (50 mg/mL, working concentration 50 µg/mL)
  • Others as needed
• Equipment:
  • Autoclave
  • Water bath (60°C)
  • Pipetman and sterile pipettes
  • Ice bucket (to store antibiotic)
  • Autoclavable flasks/bottles
  • Lab tape
  • 70% ethanol
  • Flame source
  • Antibiotic dilution reagents (e.g., sterile water, DMSO, EtOH)

Procedure

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A) From Individual Components:

1. Media Preparation:
   • For each 950 mL of MilliQ H₂O, add 10 g Tryptone, 10 g Sodium Chloride (NaCl), and 5 g Yeast Extract.
2. Mixing:
   • Stir until the powder is completely dissolved.
3. pH Adjustment:
   • Measure the pH of the solution and adjust to ~7.0 using sodium hydroxide (NaOH).
4. Final Volume Adjustment:
   • Adjust the solution to a final volume of 1000 mL with MilliQ H₂O.
5. Sterilization:
   • Transfer the solution to a heat-resistant bottle and autoclave using the liquid cycle.
6. Ampicillin Addition (if needed):
   • If adding Ampicillin, allow the LB broth to cool to ≤50°C.
   • Add 0.5 mL of Ampicillin stock solution (100 mg/mL) per 1000 mL of LB broth.
7. Storage:
   • Store the prepared LB broth at room temperature.

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B) From Pre-mixed Powder:

1. Media Preparation:
   • For each 950 mL of MilliQ H₂O, add 25 g of pre-mixed LB powder (containing Tryptone, NaCl, and Yeast Extract).
2. Mixing:
   • Stir until the powder is completely dissolved.
3. pH Adjustment:
   • Measure the pH of the solution and adjust to ~7.0 using sodium hydroxide (NaOH).
4. Final Volume Adjustment:
   • Adjust the solution to a final volume of 1000 mL with MilliQ H₂O.
5. Sterilization:
   • Transfer the solution to a heat-resistant bottle and autoclave using the liquid cycle.
6. Storage:
   • Store the prepared LB broth at room temperature.

Materials

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From Individual Components:

• Tryptone (10g)
• Sodium Chloride (NaCl) (10g)
• Yeast Extract (5g)
• MilliQ H₂O (950 mL)
• Sodium Hydroxide (NaOH) (for pH adjustment)
• Ampicillin (Amp) (optional, 100 mg/mL stock solution, 0.5 mL per 1000 mL LB Broth)

From Pre-mixed Powder:

• Pre-mixed LB powder (25g)
• MilliQ H₂O (950 mL)

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Equipment

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1. Stirring rod or magnetic stirrer
2. pH meter
3. Autoclave
4. Measuring cylinder (for final volume adjustment)
5. Heat-resistant glass bottles or flasks (for autoclaving)
6. Pipettes and tips (if adding Ampicillin)
7. Autoclave-safe container for storage

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Reagents

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1. NaOH (for pH adjustment)
2. MilliQ H₂O
3. Tryptone
4. Sodium Chloride (NaCl)
5. Yeast Extract
6. Ampicillin (optional, 100 mg/mL stock)
7. Pre-mixed LB powder (if applicable)

Procedure

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1. DNA Resuspension:

   Add 10µL of dH₂O to the selected DNA wells from the Distribution Kit. Pipette up and down several times to ensure thorough mixing, then let it sit for a few minutes. The solution should turn red due to the cresol red dye.

2. Tube Preparation:

   Label 1.5mL microcentrifuge tubes with the part names or well numbers. Pre-chill the tubes in a floating foam rack on ice.

3. Competent Cell Thawing:

   Thaw competent cells on ice for about 10-15 minutes. Discard any unused thawed cells, as refreezing will significantly reduce their efficiency.

4. Adding Competent Cells:

   Transfer 50µL of thawed competent cells into each pre-labeled and pre-chilled tube. Keep all tubes on ice during the process.

5. Adding DNA to Cells:

   Pipette 1µL of the resuspended DNA into the competent cell tubes. Gently pipette up and down to mix the solution. Keep the tubes on ice.

6. Control Transformation:

   In a separate tube with 50µL of competent cells, add 1µL of positive control DNA (10pg/µL). Gently mix by pipetting up and down. Keep this control tube on ice as well.

7. Incubation on Ice:

   Close the tubes and incubate them on ice for 30 minutes. You can gently flick the tubes occasionally to mix, but return them to the ice immediately after.

8. Heat Shock:

   Transfer the tubes to a 42°C water bath for 45 seconds. Make sure the bottoms of the tubes are fully submerged in the water. Exact timing is critical at this step.

9. Return to Ice:

   Immediately return the tubes to ice for an additional 5 minutes after heat shock.

10. Adding SOC Media:

    Add 950µL of SOC media (pre-warmed to room temperature) to each tube. SOC media should be stored at 4°C when not in use to prevent contamination.

11. Incubation:

    Incubate the tubes at 37°C for 1 hour, shaking at 200-300 rpm.

12. Plating the Transformations:

    Spread 100µL of the transformation mixture onto LB agar plates containing the appropriate antibiotic. Use a sterile spreader or glass beads to evenly distribute the mixture.

13. Optional Spin Down for Low DNA Samples:

    If necessary for low DNA samples, centrifuge the cells at 6800g for 3 minutes. Remove 800µL of the supernatant, resuspend the cells in the remaining 100µL, and plate as described above.

14. Plate Incubation:

    Place the plates agar side up and incubate them at 37°C for 14-18 hours. Avoid over-incubation, as colonies may overgrow, and the antibiotics may degrade, leading to the growth of untransformed cells.

15. Colony Selection:

    After incubation, select individual colonies for further analysis, such as colony PCR, or prepare glycerol stocks for long-term storage.

16. Control Colony Count:

    Count the colonies on the control plate to determine transformation efficiency. Competent cells should yield an efficiency between 1.5 × 10⁸ and 6 × 10⁸ cfu/µg DNA.

Materials

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• Resuspended DNA to be transformed
• 10pg/µl Positive transformation control DNA (e.g. pSB1C3 with BBa_J04450, RFP on high-copy chloramphenicol-resistant plasmid)
• Competent Cells (50µl per sample)
• SOC Media (950µL per sample)
• Petri plates with LB agar and antibiotic (2 per sample)

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Equipment

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1. Centrifuge
2. Heat block (42°C water bath)
3. Vortexer
4. Ice & ice bucket
5. Floating foam tube rack
6. Lab Timer
7. 37°C incubator
8. Sterile spreader or glass beads
9. Microcentrifuge
10. Pipettes and tips (P10, P20, P200)

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Reagents

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1. Pipettes: P20 and P200
2. gBlocks (if applicable)
3. Elution buffer (for DNA resuspension)
4. SOC media
5. Competent cell test kit (if needed)