Protocol

DNA Amplification Extraction Transformation Polymerase Chain Reaction Others DNA Recombination Gibson Assembly Restriction Enzyme Digestion Ligation Protein Assay Protein Purification SDS-Page Functional Assay Heme Staining Fluorescent Intensity and OD Measurement

DNA Amplification

Extraction

1. Genomic DNA Extraction

Day1

  1. Pick a single colony and dip the inoculation loop into 5 mL LB + appropriate antibiotics broth.
  2. Culture the bacteria for 12-16 hrs(O/N culture).

Day2

  1. Harvesting
    1. Transfer 1.5 mL of cultured bacterial cells to an Eppendorf tube.
    2. Centrifuge at 13,000 rpm for 1 Minute at room temperature.
    3. Discard the supernatant.
    4. Repeat the harvesting step until all the samples have been used out.
  2. Sample preparation
    1. Add 180 µL of GT buffer and vortex to resuspend the cell pellet.
    2. Add 8 µL proteinase K(20mg/mL).
    3. Put them in a 60°C water bath for 15 minutes.
    4. During the 15 minutes, invert every 3 minutes.
  3. Lysis
    1. Add 200 µL of GB buffer and vortex for 10 seconds.
    2. Put the eppendorf in a 70°C dry bath for 15 minutes.
    3. During the 15 minutes, invert every 3 minutes.
    4. At this time, pre-heat the ddH2O to 70°C(for Elution).
    5. After 70°C incubation, add 10 µL of RNase A(10mg/mL) to the clear lysate then shake vigorously.
    6. Incubate at room temperature for 8 minutes.
  4. DNA Binding
    1. Add 200 µL of absolute ethanol and mix immediately by shaking vigorously.
    2. Break up the precipitate with a Pipette.
    3. Place the GD column in a collection tube and transfer all the mixture into the columns.
    4. Centrifuge at 13,000 rpm for 3 minutes.
    5. Discard the flow-through and place the GD column in a new collection tube.
  5. Wash
    1. Add 400 µL W1 buffer to the column, centrifuge at 13,000 rpm for 1 minute.
    2. Discard the flow-through.
    3. Add 600 µL Wash buffer, centrifuge at 16,000 rpm for 1 min.
    4. Discard the flow-through.
    5. Centrifuge at 13,000 rpm for 5 mins to dry the column matrix.
    6. Replace the collection tube with an eppendorf.
  6. Elution
    1. Add 20 µL pre-warm ddH2O to the center of the column, stand for 3 minutes.
    2. Centrifuge at 13,000 rpm for 3 minutes..

2. Plasmid DNA Extraction

Day1

  1. Pick a single colony and dip the inoculation loop into 5 mL LB + appropriate antibiotics broth.
  2. Culture the bacteria for 12-16 hrs(O/N culture).

Day2

  1. Harvesting
    1. Transfer 1.5 mL of cultured bacterial cells to an Eppendorf tube.
    2. Centrifuge at 13,200 rpm for 1 Minute at room temperature.
    3. Discard the supernatant.
    4. Repeat the harvesting step until all the samples have been used out.
  2. Resuspension
    1. Add 200 µL of PD1 Buffer (make sure RNase A was added) to the eppendorf tube containing the cell pellet.
    2. Suspend the cell pellet completely by vortex.
  3. Cell lysis
    1. Add 200 µL of PD2 Buffer then mix gently by inverting the tube 10 times.
    2. Let stand at room temperature for at least 2 minutes, do not exceed 5 minutes.
  4. Neutralization
    1. Add 300 µL of PD3 Buffer then mix immediately by inverting the tube 10 times.
    2. Centrifuge at 13,200 rpm for 3 minutes at room temperature.
    3. During centrifugation, place a PDH Column in a 2 mL Collection tube.
  5. DNA Binding
    1. Transfer all of the supernatants to the PDH Column.
    2. Centrifuge at 13,200 rpm for 30 seconds at room temperature.
    3. Discard the flow-through.
  6. Wash
    1. Add 600 µL of Wash Buffer (with absolute ethanol) into the PDH Column.
    2. Centrifuge at 13,200 rpm for 30 seconds at room temperature.
    3. Discard the flow-through.
    4. Place the PDH Column back in the 2 mL Collection Tube.
    5. Centrifuge at 13,200 rpm for 3 minutes at room temperature to dry the column matrix.
    6. Transfer the dried PDH Column to a new eppendorf.
  7. Elution
    1. Add 50 µL of ddH2O into the center of the column matrix.
    2. Let stand for 3-5 minutes.
    3. Centrifuge at 13,200 rpm for 3 minutes at room temperature.

Use nanodrop spectrophotometer:

  1. Wash with 1 µL ddH2O.
  2. Drop 1 µL ddH2O as blank.
  3. Drop 1 µL of the sample.
  4. Measure the absorbance of 260/230 and 260/280. The ratio of absorbance is ideal at 1.8~2.0.
  5. Write down the concentration on the eppendorf.

Transformation

1. Chemically Competent Cell Preparationn

Day 1-Overnight culture

  1. Prepare two falcon tubes containing 10 mL LB.
  2. Inoculate a single colony with an inoculation loop into the falcon tube.
  3. Culture at 37°C, 200 rpm for 16-20 hrs.

Day 2- Making competent cell

  1. Two flasks of 300 mL LB are pre-warm under 37°C.
  2. Inoculate 300 mL LB with 3 mL culture in two flasks.
  3. Incubate at 37 °C, 200pm until 0D600 = 0.2-0.5 (approx. 2 hrs). Put eppendorf tubes on ice now so that they are cold when cells are aliquotted into them later.
  4. Transfer the bacteria broth into 50 mL falcon tubes and incubate on ice for 30 min.
  5. Centrifuge at 400 x g, 5 mins, 4 °C
  6. Decant the supernatant and resuspend the pellet gently in 25 mL ice-cold 0.1 M MgCl2 solution.
  7. Incubate on ice for 30 min.
  8. Centrifuge at 4000 x g, 5 min, 4 °C.
  9. Decant the supernatant and resuspend the pellet gently in 25 mL ice-cold 0.1 M CaCl2 solution.
  10. Incubate on ice for 30 min.
  11. Centrifuge at 4000 x g, 5 min, 4 °C.
  12. Decant the supernatant and resuspend the pellet gently in 5 mL ice-cold CaCl2/Glycerol solution.
  13. Freeze at -20°C.
  14. Store at - 80 °C freezer.

2. Competent Cell Transformation Efficiency Testing

  1. Clean up the working area by wiping it down with 70% ethanol.
  2. Thaw competent cells on ice. Label one 1.5 mL microcentrifuge tube for each transformation and then pre-chill by placing the tubes on ice. Do triplicates (3 each) of each concentration if possible, so you can calculate an average colony yield.
  3. Spin down the DNA tubes containing your controls to collect all of the DNA into the bottom of each tube prior to use. A quick spin of 20-30 seconds at 8,000-10,000 rpm will be sufficient.
  4. Pipet 1 µL of DNA into each microcentrifuge tube.
  5. Pipet 50 µL of competent cells into each tube. Flick the tube gently with your finger to mix.
  6. Incubate on ice for 30 minutes. Pre-heat water bath now to 42°C.
  7. Heat-shock the cells by placing them into the water bath for 1 minute. Be careful to keep the lids of the tubes above the water level, and keep the ice close by.
  8. Immediately transfer the tubes back to ice, and incubate on ice for 5 minutes.
  9. Add 250 µL of LB per tube, and incubate at 37°C for 1 hour shaking at 200 rpm.
  10. Pipet 30 µL from each tube onto the appropriate plate, and spread the mixture evenly across the plate. Incubate at 37°C overnight or approximately 16 hours. Position the plates with the agar side at the top, and the lid at the bottom.
  11. Count the number of colonies on a light field or a dark background, such as a lab bench. Use the following equation to calculate your competent cell efficiency. If you've done triplicates of each sample, use the average cell colony count in the calculation.
    • Efficiency (in cfu/µg) = [colonies on plate (cfu) / Amount of DNA plated (ng)] x 1000 (ng/µg)
    • Note: The measurement "Amount of DNA plated" refers to how much DNA was plated onto each agar plate, not the total amount of DNA used per transformation. You can calculate this number using the following equation:
    • Amount of DNA plated (ng) = Volume DNA added (1 µL) x concentration of DNA (refer to vial, convert to ng/µL) x [volume plated (100 µL) / total reaction volume (1000 µL)]

3. Transformation

  1. Pre-heat water bath to 42°C.
  2. Prepare the ice with water.
  3. Put the sterile Eppendorf tubes on the ice.
  4. Make sure the plasmid is ready.
  5. Take competent cells out of -80°C and thaw on ice immediately.
  6. Mix 1-5 µL of plasmid DNA with 30-35 µL of competent cells. Gently mix by flicking the bottom of the tube. (Do not vortex!)
  7. Incubate the competent cell and DNA mixture on ice for 30 mins.
  8. Heat shock in a 42°C water bath for 1 minute.
  9. Put the tubes back on ice for 5 min.
  10. Plate the transformation onto an LB agar plate with a selection marker.
  11. Incubate plates at 37°C overnight.
  12. The next day, observe the growth of bacteria colonies.

Polymerase Chain Reaction

1. PCR

...

Program setup:

  1. Initial Denaturation 98°C / 3min
  2. Denaturation 98°C / 30sec
  3. Primer annealing 56°C / 30 sec
  4. Extension 70°C / 5 min
  5. Repeat from steps 2 to 4 for 30-35 cycles.
  6. Final extension 70°C / 15 min

2. PCR Cleanup

  1. Sample Preparation
    1. Transfer up to 300 μL of reaction product to a 1.5 mL microcentrifuge tube.
    2. Add 1,300 μL of Gel/PCR Buffer to the sample and mix by vortex.
    3. If the color of the mixture has turned purple, add 10 μL of 3M Sodium Acetate (pH5.0) and mix thoroughly.
  2. DNA Binding
    1. Place a DFH Column in a 2 mL Collection Tube.
    2. Transfer the sample mixture to the DFH Column.
    3. Centrifuge at 14-16,000 x g for 30 seconds.
    4. Discard the flow-through.
    5. Place the DFH Column back in the 2 mL Collection Tube.
  3. Wash
    1. Add 600 μL of Wash Buffer (make sure ethanol was added) into the center of the DFH Column.
    2. Let stand for 1 minute at room temperature. Centrifuge at 14-16,000 x g for 30 seconds.
    3. Discard the flow-through and place the DFH Column back in the 2 mL Collection Tube.
    4. Centrifuge for 3 minutes at 14-16,000 x g to dry the column.
  4. DNA Elution
    1. Transfer the dried DFH Column to a new 1.5 mL microcentrifuge tube.
    2. Add 20 μL of (60-70°C) pre-heated ddH2O into the center of the column matrix.
    3. Let stand for at least 2 minutes to ensure ddH2O is completely absorbed.
    4. Centrifuge for 2 minutes at 14-16,000 x g to elute the purified DNA.

Others

1. Electrophoresis

  1. Preparation (1-2% agarose gel)
    • Large: 50 mL TAE buffer + 0.5-1.0 g agarose
    • Small: 30 mL TAE buffer + 0.3-0.6 g agarose
  2. 50X TAE Buffer dilution(100 mL)
    1. Add 50 mL of 1X TAE Buffer into a 125 mL Erlenmeyer flask.
    2. Add 0.5 g of agarose.
    3. Microwave for 1-3 min until the agarose is completely dissolved.
    4. Add 2.5 µL of safety dye.
    5. Wait until it cools down to about 50°C.
    6. Pour the agarose into a gel tray and place the well comb in place.
  3. Making gel
    1. Add 200 µL of GB buffer and vortex for 10 seconds.
    2. Put the eppendorf in a 70°C dry bath for 15 minutes.
    3. During the 15 minutes, invert every 3 minutes.
    4. At this time, pre-heat the ddH2O to 70°C(for Elution).
    5. After 70°C incubation, add 10 µL of RNase A(10mg/mL) to the clear lysate then shake vigorously.
    6. Incubate at room temperature for 8 minutes.
  4. Run electrophoresis
    1. Once solidified, place the gel and pour the TAE Buffer into the electrophoresis chamber.
    2. Add 4 μL loading dye to a 20 μL sample for a big well or add 2 μL loading dye to a 10 μL sample for a small well.
    3. Load an appropriate marker and samples into the well.
    4. Run 70 V, for at least 30 min.

2. Gel Extraction

  1. Gel Dissociation
    1. Excise the agarose gel slice containing relevant DNA fragments and remove any extra agarose to minimize the size of the gel slice.
    2. Transfer up to 300 mg of the gel slice to a 1.5 mL microcentrifuge tube.
    3. Add 500 μL of Gel/PCR Buffer and mix by vortex.
    4. Incubate at 55-60°C for 10-15 minutes to ensure the gel slice has been completely dissolved.
    5. During incubation, invert the tube every 2-3 minutes.
    6. If the color of the mixture has turned purple, add 10 μL of 3M Sodium Acetate (pH5.0) and mix thoroughly.
    7. Cool the dissolved sample to room temperature.
  2. DNA Binding
    1. Place a DFH Column in a 2 mL Collection Tube.
    2. Transfer 800 μL of the sample mixture to the DFH Column.
    3. Centrifuge at 14-16,000 x g for 30 seconds.
    4. Discard the flow-through and place the DFH Column back in the 2 mL Collection Tube.
    5. If the sample mixture is more than 800 μL, repeat the DNA Binding step.
  3. Wash
    1. Add 400 μL of W1 Buffer into the DFH Column.
    2. Centrifuge at 14-16,000 x g for 30 seconds then discard the flow-through.
    3. Place the DFH Column back in the 2 ml Collection Tube.
    4. Add 600 μl of Wash Buffer (make sure ethanol was added) into the DFH Column.
    5. Let stand for 1 minute at room temperature. Centrifuge at 14-16,000 x g for 30 seconds then discard the flow-through.
    6. Place the DFH Column back in the 2 mL Collection Tube.
    7. Centrifuge at 14-16,000 x g for 3 minutes to dry the column matrix.
  4. DNA Elution
    1. Transfer the dried DFH Column to a new 1.5 mL microcentrifuge tube.
    2. Add 20 μL of (60-70°C) pre-heated ddH2O into the center of the column matrix.
    3. Let stand for at least 2 minutes to ensure ddH2O is completely absorbed.
    4. Centrifuge for 2 minutes at 14-16,000 x g to elute the purified DNA.

DNA Recombination

Gibson Assembly

...

Steps:

  1. Add the components in the following order: ddH2O, 2X Gibson Assembly master mix, and DNA fragments.
  2. Use a PCR machine to incubate at 50°C for 15 min.
  3. Perform the transformation immediately.

Restriction Enzyme Digestion

...

Steps:

  1. Prepare the sterilized eppendorfs.
  2. Add the component above in the following order. ddH2O, 10X fast digest buffer, DNA, restriction enzyme.
  3. Mix the components by pipetting.
  4. Put it in the 37°C water bath for an hour.

Ligation

...

Steps:

  1. Combine the components in a PCR or Eppendorf tube in the following order: ddH2O, DNA, T4 DNA ligase buffer, T4 DNA ligase
  2. Incubate at room temperature for 2 hours or at 4°C overnight.

Protein Assay

Protein Purification(Ni-Column)

Day 1-Growth of expression cultures

  1. Inoculate 5 mL of LB medium containing the appropriate antibiotics with a fresh bacterial colony harboring the expression plasmid.
  2. Grow at 37°C overnight.li>

Day 2-Purification

  1. Harvest the cells by centrifugation at 16,000 rpm for 2 min for 5 times.
  2. Resuspend a pellet derived from 5 mL cell culture volume in 630 uL Lysis Buffer (NPI-10).
  3. Add 70 ul Lysozyme Stock Solution (10 mg/mL) and add 0.6 uL Benzonase Nuclease(25 Units/uL).
  4. Incubate on ice for 30 min
  5. Centrifuge lysate at 16,000 rpm for 20 min at 4°C. Collect supernatant. Save 20 uL of the cleared lysate for SDS-PAGE analysis.
  6. Equilibrate the Ni-NTA spin column with 600 uL Buffer NPI-10.
  7. Centrifuge for 2 min at 3,150 rpm.
  8. Load 600 uL of the cleared lysate containing 6X His-tagged protein onto the pre-equilibrated Ni-NTA column.
  9. Centrifuge for 5 min at 1,600 rpm. Save the flow-through for analysis by SDS-PAGE to check binding efficiency.
  10. Wash the Ni-NTA spin column twice with 600 uL Buffer NPI-20.
  11. Centrifuge for 2 min at 2,900 rpm. Save the flow-through (wash fractions) for analysis by SDS-PAGE to check the stringency of the wash conditions.
  12. Elute the protein twice with 300 uL Buffer NPI-500.
  13. Centrifuge for 2 min at 2,900 rpm and collect the elute.

SDS-Page

...

Steps:

  1. Discard the overlayed water on the resolving gel.
  2. Add the 4% stacking gel solution until it overflows. Insert the comb immediately ensuring no air bubbles are trapped in the gel or near the wells.
  3. Allow the gel to set for about 20-30 min at room temperature.
  4. Place the gel in a clean plastic electrophoresis chamber and the corresponding gel holder.
  5. Fill the inner portion between the gel(s) and the gel holder with the appropriate 1X Running Buffer. Pour the remaining 1X Running Buffer into the outer chamber.li
  6. Load the Ladder and the samples with 4X protein loading dye into the wells.
  7. Allow the gel to electrophorese for 45–90 minutes. Turn OFF the power immediately after the dye front migrates out from the bottom of the gel.

Functional Assay

Heme Staining

  1. After running SDS-Page, rinse the gel with water for 5 minutes.
  2. Immediately before use, mix 30mL TMBZ solution with 70mL sodium acetate solution (per gel).
  3. Immerse the gel in the solution above. put gel in the dark.
  4. Mix occasionally (every 15 minutes) for 1-2 hours
  5. Add 495 μL 30% hydrogen peroxide (final concentration 30 mM in 100 mL). Mix well.
  6. Staining should be visible within 3 minutes and increase in intensity over the next 30 minutes.
  7. The background of the gel may be removed by destaining with 3:7 isopropanol:0.25M sodium acetate. This mixture can be replaced once or twice with a fresh solution. Gels may also be stored in this solution.
  8. Place the gel in a plastic sheet protector.
  9. Place gel in the scanner.

Fluorescent Intensity and OD Measurement

  1. Incubate E.coli with mCerulean gene in LB+CM liquid and incubate E.coli DH5a in LB liquid for about 12 hours.
  2. Collect the overnight culture in separate eppendorf by centrifugation
  3. Wash the pellet with 1xPBS. After centrifugation, discard the supernatant. Use pipetman to remove any remain liquid.
  4. Add 1ml of LB liquid and LB+CM liquid to each eppendorf, vortex, and take 500ul from each for measuring OD600
  5. Add 200ul of E.coli with mCerulean gene culture to flasks containing LB+CM and add the same quantity of DH5a to flasks containing LB
  6. Measure OD600 (1x, 1/2x, 1/4x dilutions) and fluorescent density (1x, 1/2x, 1/4x dilutions), and take photographs.
  7. Place in the incubator, start the shaker, and wait for the next time period.
... ...

Contact

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No. 155, Section 2, Linong St, Beitou District, Taipei City, 112
+886-2-2826-7000
nycu.taipei.igem@gmail.com

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