Team:PuiChing Macau/Experiments

Protocols

Material List

1. PCR

  • New England BioLabs Q5® Hot Start High-Fidelity 2X Master Mix. Catalog number: M0494S

2. Gel electrophoresis

  • Bio Rad 50x TAE Buffer Catalog number: 166-0742
  • Bio Rad Certified™ Molecular Biology Agarose
  • Invitrogen SYBR® Safe DNA gel stain. Catalog number: S33102
  • New England BioLabs Gel Loading Dye Purple (6x). Catalog number: B7024S
  • Invitrogen 1 Kb Plus DNA Ladder. Catalog number: 10787018

3. DNA and Gel Purification

  • illustra™ GFX™ PCR DNA and Gel Band Purification Kit

4. DNA Assembly

  • New England BioLabs NEBuilder® HiFi DNA Assembly Master Mix. Catalog number: M5520AA

5. Transformation of Escherichia coli

6. Miniprep plasmid purification

  • QIAprep® Spin Miniprep Kit

7. Exfection™ Plasmid EF midi

  • GeneAll® Exfection™ Plasmid EF midi

8. Blue-Green Medium (BG11) preparation

9. Nitrocellulose Membrane preparation

  • Bio Rad Nitrocellulose Membrane, 0.2um

10. Transformation of Cyanobacteria (Synechococcus sp.)

  • SIGMA Spectinomycin dihydrochloride penta-hydrate

11. Western bolt

Experiment Protocol

1. PCR

　
1x
forward primer 2.5µL
reverse primer 2.5µL
Template 1µL
H2O 19µL
NEB Q5® Hot Start High-Fidelity 2X Master Mix 25µL
　
step 1 step 2 (35cycles) step 3 step 4
initial denaturation denaturation anneal extension final extension stock
98°C 98°C 55°C 72°C 72°C 4°C
3mins 10s 30s * 2mins ∾

*30 seconds/kb

2. Gel electrophoresis

1.Gel preparation

1x
Bio Rad TAE Buffer 30mL
Bio Rad Certified™ Molecular Biology Agarose *
Invitrogen SYBR® Safe DNA gel stain **

*depend on the desired gel concentration

**1µL SYBR® Safe per 1mL TAE Buffer

2.Sample loading

DNA sample : Gel Loading Dye Purple (6x) =5:1

3. DNA and Gel Purification

  1. Cut gel and measure the weight.
  2. Add Capture Buffer 3 (10µL/10mg), shake and heat at 60°C, 1000rpm for 10mins with ThermoMixer.
  3. Transfer 750µL mixture to a microspin column, centrifuge at 16000×g for 30s.
  4. Add 600µL Wash Buffer 1 and centrifuge at 16000×g for 30s twice.
  5. Add 50µL Elution Buffer and centrifuge at 16000×g for 30s.

4. DNA Assembly

  1. Calculations

    2. Calculation rules: Vector:Insert=1:2 (in pmol)

    Total DNA amount: 0.03pmol~0.2pmol

    Total DNA volume<=10µL

    pmol=ng*1000/(bp*650)

  2. According to the calculations, mix the DNA samples and 10µL of NEBuilder® HiFi DNA Assembly Master Mix M5520AA.
  3. Put the mixture at 50°C for 15 mins.

5. Transformation of Escherichia coli

  1. Add the assembled product to 100µL of competent cells (Escherichia coli), place on ice for 20mins.
  2. Heat shock at 42°C for 1min.
  3. Transfer to ice for 2mins.
  4. Add 900µL of LB to the tube, incubate at 37°C for 1 hour. Shake at 250rpm.
  5. Warm the LB agar plate with antibiotics* to 37°C.
  6. Centrifuge at 12000rpm for 30s. Concentrate the cells to 100µL.
  7. Spread 100µL cells onto the plate.
  8. Incubate overnight at 37°C.

*antibiotics (spectinomycin/ampicillin/cam): 50µg/mL

6. Miniprep plasmid purification

  1. Pellet 1.5mL bacteria each tube and centrifuge at 6800×g for 3mins at room temperature for 3 times.
  2. Resuspend pelleted bacteria in 250µL Buffer P1.
  3. Add 250µL Buffer P2 and mix 6 times.
  4. Add 350µL Buffer N3 and mix 6 times.
  5. Centrifuge for 10mins at 13000rpm.
  6. Apply 800µL supernatant to the column, centrifuge for 30s.
  7. Add 0.5mL Buffer PB and centrifuge for 30s.
  8. Add 0.75mL Buffer PE and centrifuge for 60s.
  9. Centrifuge for 1min.
  10. Add 30µL Buffer EB and stand for 1min. Centrifuge for 1min. Stock at -20°C.

7. Exfection™ Plasmid EF midi

  1. Pellet 200mL (50mL each tube) of bacterial culture by centrifugation for 10mins at 5000×g in a tabletop centrifuge. Discard the supernatant as much as possible.
  2. Resuspend pelleted bacterial cells thoroughly in 4mL of Buffer P1. Combine into 2 tubes.
  3. Add 4mL of Buffer P2 and mix by inverting the tube 5~6 times (do not vortex). Incubate until the cell suspension becomes clear. Do not incubate for more than 5mins.
  4. Add 4mL of Buffer P3 and thoroughly but gently mix by inverting the tube 7~8 times (do not vortex).
  5. Centrifuge at 4500×g for 10mins, then transfer the supernatant into Ezclear™ filter (blue ring) sitting on a 50mL conical collection tube (provided). Let it stand for 2mins and centrifuge for 2mins at 1500×g (2800rpm).
  6. Apply 500µL of Buffer ER to the filtrate and close the cap of 50mL conical tube.
  7. Vortex to mix and incubate for 15mins on ice.
  8. Incubate for 15mins at 37ºC and centrifuge for 2mins at 1500×g (2800rpm).
  9. Transfer carefully the upper phase (clear) into a fresh 15mL conical tube (provided) by pipetting.
  10. Add ½ volume of Buffer EG to the transfer and invert several times to mix completely.
  11. Transfer all the mixture to SV midi column (clear ring) by decanting or pipetting. Centrifuge for 2mins at 1500×g. Remove the column, discard the pass-through, and re-insert the column to the collection tube.
  12. Apply 10mL of Buffer EW1, centrifuge for 2mins at 1500×g. Remove the column, discard the pass-through and re-insert the column to the collection tube.
  13. Apply 10mL of Buffer EW2, centrifuge for 2mins at 1500×g. Remove the column, discard the pass-through, and re-insert the column to the collection tube.
  14. Apply 3mL of Buffer EW2, centrifuge for 15mins at 4500×g. Transfer the column to a new 50mL conical tube (provided).
  15. Add 0.6mL of Buffer EF directly onto the centre of the column membrane and close the cap. Incubate for 5mins at room temperature and centrifuge for 5mins at 4500×g.

8. Blue-Green Medium (BG11) preparation

  1. Stocks
    2. per 500mL
    1 NaNO3 75.0g
    per 500mL
    2 H2HPO4 2.0g
    3 MgSO4•7H2O 3.75g
    4 CaCl2•2H2O 1.80g
    5 Citric Acid 0.30g
    6 Ammonium ferric citrate green 0.30g
    7 EDTA Na2 0.05g
    8 Na2CO3 1.00g
    9
    Trace metal solution per liter
    H3BO3 2.86g
    MnCl2•4H2O 1.81g
    ZnSO4•7H2O 0.22g
    Na2MoO4•2H2O 0.39g
    CuSO4•5H2O 0.08g
    Co(NO3)2•6H2O 0.05g
  2. Medium
    3. per liter
    Stock solution 1-810.0mL
    Stock solution 91.0mL
  3. Agar
    4. per liter
    BG11 medium1000mL
    Agarose25g

9. Nitrocellulose Membrane preparation

  1. Cut the membrane into circles.
  2. Soak the membranes into distilled water for 5mins and pour it out. Repeat this step for three times.
  3. Soak the membranes into distilled water overnight.
  4. Heat the membranes using a microwave oven for 3mins and autoclave.

10. Transformation of Cyanobacteria (Synechococcus sp.)

  1. Collect 15 mL cell (OD730=0.8-1.0) from the conical flask.
  2. Centrifuge at 4000rpm for 10mins, discard the supernatant.
  3. Using the same volume of BG11 medium to resuspend the cells and centrifuge at 4000rpm for 10mins again to wash the cells.
  4. Separate the cells into 200µL portions, add plasmids (conc. 10µg/mL) to each portion. Light incubate for 4 hours.
  5. Spread 100µL of the mix on the nitrocellulose membranes which are soaked in BG11 agar plates without antibiotics. Light incubate overnight (for 18-20 hours).
  6. Transfer the nitrocellulose membranes with the cells to a new BG11 agar plate containing antibiotics*. Light incubate for 7-10 days until the single colonies appear. Check periodically for the growth condition and water condensation to prevent other bacteria growing on the plate.
  7. Take a single colony, spread it on a new BG11 agar plate containing antibiotics and incubate under light.
  8. Extract the cells growing on the new BG11 agar plate, culture in fresh BG11 medium containing antibiotics for 4 days (until logarithmic phase).
  9. PCR validation: Pellet 1-2mL of cells to a microcentrifuge tube, lysis the cells by shaking with quartz sands to extract the DNA. Take 0.5µL of the DNA as a template to conduct PCR and take 3µL of the PCR product to conduct gel electrophoresis. Use the DNA of wild type Cyanobacteria as a control.

*spectinomycin (10µg/mL)

11. Protein extraction

  1. Pellet 100mL of bacterial culture by centrifugation for 10mins at 6000×g in a tabletop centrifuge. Discard the supernatant as much as possible.
  2. Resuspend pelleted bacterial cells in 250µL of 50M HEPES.
  3. Microtip sonication: 40%AMPL, 45s processing, 1s on-off. (3 times)
  4. Centrifuge at 1200×g for 10mins.
  5. Transfer the supernatant to a microspin tube, stock at -20ºC.

12. Western bolt

  1. Protein processing: the extracted proteins were added into 4xloading, then boiled in water for 10min
  2. Sds page preparing and runing: 12% sds-gel refer to SDS gel preparation.
  3. Transfer the gel into PDFV membrane: refer to Transfer membrane.
  4. Antibody incubation : primary antibody : anti-His rabbit developed(1:2000 diluted); second antibody: anti-rabbit conjugated HRP(1:5000 diluted) refer to Antibiotic staining.

13. SDS gel preparation

  1. 10% resolving gel solution
    2. 1x
    SureCast™ Acrylamide (40%)2.0mL
    SureCast™ Resolving Buffer2.0mL
    Distilled water3.9mL
    10% SureCast™ APS80µL
    Total8mL
  2. Add 8 μL of SureCast™ TEMED for every 8 mL of resolving gel solution. Mix well (but gently) and proceed immediately to Pour resolving gel.
  3. Tilt the handcast station to recline on the heel.
  4. Add resolving gel solution to the glass plates until the solution reaches the level of the fill line.
  5. Carefully overlay the resolving gel solution with butanol, or isopropanol.
  6. Set the handcast station back to the upright position.
  7. Allow the resolving gel to polymerize (5–10 min). The interface becomes more distinct as the gel polymerizes.
  8. Verify polymerization by examining left over acrylamide in the tube.
  9. If overlay was used, pour off the overlay solution and rinse with water (dispose of waste in the appropriate manner). Wick out any remaining liquid with a piece of blotting paper, and proceed to Prepare stacking gel solution.
  10. 4% stacking gel solution
    11. 1x
    SureCast™ Acrylamide (40%)0.30mL
    SureCast™ Staking Buffer0.75mL
    Distilled water1.92mL
    10% SureCast™ APS30µL
    Total3mL
  11. Add 3 μL of SureCast TEMED for every 3 mL of resolving gel solution. Mix well and proceed immediately to Pour stacking gel.
  12. Tilt the handcast station to recline on the heel.
  13. Add the stacking gel solution until it reaches the upper edge of the front glass plate.
  14. Set the handcast station back to the upright position.
  15. Insert the comb slowly by starting at one end and sliding it between the glass plates until both ends are in place.
  16. Allow the stacking gel to polymerize (5–10 min).
  17. Verify polymerization by examining left over acrylamide in the tube.
  18. Release the clamp and remove the plate. The gel can be used immediately, or wrapped in a damp paper towel and stored at 4°C for future use.

14. Transfer membrane

  1. Place the cathode core (–) on a flat surface.
  2. Add 5–10 mL of 1X Transfer Buffer to the cathode core (–).
  3. Place a soaked sponge pad into the cathode core (–) of the blot module. Remove any bubbles with the Blotting Roller.
  4. Take the gel with filter paper (from “Prepare gel for transfer” step 11, page 12) and place it on top of the sponge pad, with the gel side on top.
  5. Wet the surface of the gel with 1X Transfer Buffer and remove any bubbles with the Blotting Roller.
  6. Place a pre-soaked transfer membrane on the gel. Remove any bubbles with the Blotting Roller.
  7. Wet a piece of filter paper in 1X Transfer Buffer and place it on top of the transfer membrane. Remove any bubbles with the Blotting Roller.
  8. Place a pre-soaked sponge pad onto the filter paper. Remove any bubbles with the Blotting Roller.
  9. Complete the module assembly by placing the anode core (+) on top of the assembled blot sandwich.
  10. Press the two module halves together.
  11. Snap the electrophoresis tank into the base. Make sure any Cassette Clamps are removed from the chambers.
  12. Insert the blot module with the cathode core (–) facing the front. The blot module should be seated so that the electrodes clips of the module make contact with the electrode bar of the electrophoresis tank.
  13. If necessary, add 1X Transfer Buffer to the module core to completely submerge the blot sandwich. Note: do not fill above the level of the gasket in the blot module.
  14. Make sure the power supply is off and place the cover on the tank. Push all the way down so that the cover is firmly in place.
  15. Plug the power leads into your power supply.
  16. Turn on the power supply, program settings (see “Transfer Conditions”) as needed, and start transfer.
  17. After protein transfer is complete, turn the power supply off, unplug the power leads, and remove the lid.
  18. Remove the blot module from the chamber and empty the transfer buffer into an appropriate hazardous waste disposal container.
  19. Open the module assembly.
  20. Disassemble the blot sandwich and carefully remove the membrane with Blotting Tweezers.
  21. Wash the membrane using 20 mL of ultra-pure water for 5 minutes, two times.

15. Antibiotic staining

  1. Block the membrane for 1 h at room temperature or overnight at 4°C using blocking buffer.
  2. Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer. We recommend overnight incubation at 4°C; other conditions can be optimized.
  3. Wash the membrane in three washes of TBST, 5 min each.
  4. Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer at room temperature for 1 h.
  5. Wash the membrane in three washes of TBST, 5 min each.
  6. For signal development, follow the kit manufacturer’s recommendations. Remove excess reagent and cover the membrane in transparent plastic wrap.
  7. Acquire image using darkroom development techniques for chemiluminescence, or normal image scanning methods for colorimetric detection.

16. Dye decolorization

  1. Without ABTS:
    2. 1x
    BR Buffer*20µL
    500mg/L Dye**4µL
    Sample***33.4µL
    50mM HEPES142.6µL

    With ABTS:

    1x
    BR Buffer20µL
    500mg/L Dye4µL
    Sample33.4µL
    100mM ABTS2µL
    50mM HEPES140.6µL

    *pH=5

    **Coomassie Blue, Congo Red, Bromophenol Blue, Indigo Carmine

    ***3µg/µL total protein/50mM HEPES (blank of protein)/LB (blank of supernatant)

  2. Apply the configured mixture to a 96-well plate with three identical replicates for each experimental group.
  3. Measure the optical density* of each well.
  4. Seal the 96-well plate and incubate at 37ºC. Remeasure the optical density every 24 hours.

    5. *Coomassie Blue:465nm, 595nm; Congo Red: 488nm; Bromophenol Blue: 593nm; Indigo Carmine: 610nm.

17. Nickel pull down

  1. Make up 50mL of the following binding buffer: 20mM Tris 7.5, 150mM NaCl, 1% Triton-X100.
  2. Thaw 20mL medium on ice.
  3. Prepare nickel resin
  4. Pipette 500μL medium to resuspend the resin, and pipette all into the 20mL medium.
  5. Incubate and rotate it at 4°C for 45 min.
  6. Spin at 3900 rpm for 10min, remove supernatant carefully (store this, just in case!), leave ~ 1mL fraction to resuspend the resin, transfer resin to a 1.5mL tube. 4000g, 2min spin down the resin, decant the supernatant.
  7. Wash the resin with 750uL binding buffer (X3) (Note: don’t try to remove all the flow through or buffer in every wash, you will lose resin that way, just in the very last.
  8. wash try and removes as much as buffer as possible.
  9. Add 30μL 2X SDS dye, 98°C, 10min.

18. EDC degradation

  1. 1x
    sodium citrate buffer (pH=5)500µL
    3µg/µL protein sample / Ni-pulldown product500µL
    200µM ABTS1.6µL
    100µM Beta-estradiol0.8µL
  2. Shake the mixture above at 25°C for 36hr.
  3. Mix 100µL of the mixture with 400µL Acetonitrile. Centrifuge the sample at >10000rpm for 5mins to remove precipitated protein.
  4. Transfer the supernatant to another tube and dry it by nitrogen blow.
  5. Re-dissolve the dried sample in an appropriate volume (depends on concentration of targeted molecule in your sample) of acetonitrile by vivid vortex.
  6. Centrifuge the sample at >10000rpm for 5mins to remove insoluble salt. The supernatant is then ready for LCMS analysis.