Team:Hong Kong-CUHK/Protocol



Below are the protocols that we used in our project. Click the boxes for more detail!


  1. Set up PCR mixture as follow :
    2. Component Volume
    Phusion DNA Polymerase 0.5 μl
    5X Phusion Green HF Buffer 10 μl
    10mM dNTPs 1 μl
    Forward Primer 2.5 μl
    Reverse Primer 2.5 μl
    DNA template 1 μl
    DNase-free ddH2O 32.5μl
  2. Set up thermocycler for PCR reaction as follow:
    3. Procedures Temperature (°C) Duration Cycle
    Initial denaturation 98 30 s 1
    Denaturation 98 5 s 1
    Annealing 64 30 s 25
    Final extension 72 5 mins 1
    Sample keping 4 ∞ 1
  3. Add the other Component in the tube. If the added volume is less, pipetting up and down is necessary.
  4. Mix reagents in tubes by pipetting the solution up and down slowly.
  5. Quick spin the PCR tube to ensure the mixture is in the bottom of the tube.
  6. Put it in the thermocycler (PCR machine) and set the cycle information.
  7. Start the cycle and wait till it is finished.
Click here for the manufacturer's maual

  1. Prepare your 1% gel by using 0.5g of agarose in 50 ml of TAE buffer.
  2. Add 2.5ul of Ethidium Bromide before you pour your gel into the chamber.
  3. Mix 5ul of DNA with 1ul of loading buffer by pipetting up and down a couple of times.
  4. Load your samples and appropriate marker into your wells
  5. Incubate at 37°C and 250 rpm for 60-120 minutes.
  6. Apply 130 volts to the chamber for 20 mins.
  7. Check your gel using a transilluminator or other UV emitting device.
  8. Re-suspend cells by light vortexing
  9. Plate resuspended cells as above
  10. Incubate overnight at 37°C with plates upside down.
Click here for the manufacturer's maual
  1. Excise the DNA fragment from the agarose gel with a clean, sharp scalpel. Minimize the size of the gel slice by trimming off extra agarose gel.
  2. Weigh the gel slice in a colorless tube. Add 3 volumes of Buffer QG to 1 volume of gel (300 μl QG to 100 mg gel).
  3. Incubate the tube at 60°C until the gel completely dissolved.
  4. For DNA fragments <500 bp and >4 kb, add 1 gel volume of isopropanol to the sample and mix the reagents well.
  5. o bind DNA, apply 800 μl sample to the QIAquick column, and centrifuge for 1 min.
  6. Discard flow-through and place QIAquick column back in the same collection tube. It can be reused to reduce plastic waste.
  7. Add 0.75 ml of Buffer PE to wash the QIAquick column, and centrifuge for 1 min.
  8. Add 30 μl of distilled water to elute DNA.
Click here for the manufacturer's maual
  1. Prepare the reagents as follow:
    2. Reaction Components Volume
    DNA template 0.5 μg
    10X Buffer 2.5 μl
    Enzyme 2 μl
    Nuclease-Free Water to final colume of: 25 μl
  2. Pipette the solution up and down to ensure all reagents are mixed well.
  3. Place the reaction mixture at 37 oC incubation or dry bath for 2-4 hours.
  4. Purify the DNA by PCR purification kit/gel extraction kit for downstream process.
Click here for the manufacturer's maual

  1. Prepare the reaction mixture as follow:
    2. Reaction Components Volume
    DNA 0.2M KCl/HCl
    DNA template 10-200 ng
    10XT4 DNA Ligase Reaction Buffer 2 μl
    T4 DNA Ligase 1 μl
    Water Top up to 20 μl
  2. Allow the ligation to take place a 22-25°C for 10 minutes.
  3. Send 5 μl of the ligated sample should be used for agarose gel electrophoresis to confirm whether ligation has occurred (Optional)
Click here for the manufacturer's maual

  1. Prepare the Ca/glycerol buffer as follow and flow through 0.22 μM filter.
    2. Composition Volume
    0.6 M CaCl2 10 ml
    0.5 M PIPES pH7.0 2 ml
    Glycerol 15 ml
    H2O 73 ml
    Total 100 ml
  2. Spread the bacterial cell (DH5α) for plasmid replication on the LB.
  3. Pick single colony in 5 ml LB culture medium and grow overnight at 37 °C by shaking at ~220 rpm.
  4. Add 1ml overnight culture each to two of 100 ml flasks and grow the cell culture to achieve OD 600 = 0.25-0.4 (~ 3 h)
  5. Transfer the cell culture (total 200 ml) to four of 50 ml sterile centrifugation tubes.
  6. Collect the cell by centrifuging at 1000 g for 10 min at 4°C.
  7. Gently resuspend the cell in each tube with 10 ml ice-old Ca/glycerol buffer. Keep the solution ice-cold. * Cells must remain clod for the rest of the procedures!
  8. Collect the cell by centrifuging at 1000 g for 10 min at 4 °C.
  9. Centrifuge at 13,000 rpm for 1 min.
  10. Gently resuspend the cell in each tube with 1.25 ml ice-old Ca/glycerol buffer.
  11. Centrifuge at 13,000 rpm for 1 min.
  12. Column dring with centrifuge 13.000rpm for 1 min.
  13. Transfer all cells to one tube.
  14. Dispense 100 μl aliquots of competent cells into each Eppendorf.
  15. Store at -80 °C.
  16. Test the transformation efficiency of competent cells with antibiotics resistance plamids at different concentrations.
Click here for the manufacturer's maual

  1. Thaw 100µL competent E. coli cells on ice for 10 minutes
  2. Add:
    • 10-20 µl DNA from a ligation reaction mix OR
    • 50-100ng DNA of a known plasmid
  3. Place the mixture on ice for 15 minutes.
  4. Heat shock at exactly 42°C for 90 seconds.
  5. Place on ice for 5 minutes.
  6. Pipette 900 µl of room temperature SOC or LB media into the mixture.
  7. Incubate at 37°C and 250 rpm for 60-120 minutes.
  8. Pellet cells at 13000rpm for 1 minutes
  9. Remove and dispense 600 µl of supernatant
  10. Re-suspend cells by light vortexing
  11. Plate resuspended cells as above
  12. Incubate overnight at 37°C with plates upside down.
Click here for the manufacturer's maual
  1. Harvest 3 mL cells at 1OD.
  2. Centrifuge at 14,000 rpm for 30 s.
  3. Discard the supernatant and resuspend the pellet with remnant.
  4. Resuspend with 250 μl Resuspension Buffer.
  5. Add 250 μl Lysis Buffer.
  6. Add 350 μl Neutralization Buffer.
  7. Centrifuge at 13,000 rpm for 10 mins.
  8. Transfer supernatent to column.
  9. Centrifuge at 13,000 rpm for 1 min.
  10. Add 700 μl Washing Buffer B.
  11. Centrifuge at 13,000 rpm for 1 min.
  12. Column dring with centrifuge 13.000rpm for 1 min.
  13. Inoculate with 30 μl distilled water.
  14. Centrifuge at 13,000 rpm for 1 min.
Click here for the manufacturer's maual

LB Broth Formulation per one liter
  • 10 g SELECT Peptone 140
  • 5 g SELECT Yeast Extract
  • 5 g Sodium Chloride
LB Agar Formulation per one liter
  • 10 g SELECT Peptone 140
  • 5 g SELECT Yeast Extract
  • 5 g Sodium Chloride
  • 12 g SELECT Agar
2XYT Formulation per one liter
  • 16.0 gTryptone
  • 10 g Yeast Extract
  • 5 g Sodium Chloride
    • Final pH 6.8 ± 0.2 at 25°C
Click here for the manufacturer's maual
  1. Set up the following reactions:
    2. Component Volume
    DNA template ≤2 μg
    Amino a cid Mixture (-Met) 5 μl
    S30 Pewmix without amino acids 20 μl
    [35S]Methionine 1 μl
    Reverse Primer 2.5 μl
    S30 Extract, Circular 15 μl
    Nucelase-Free Water to final volume of : 50 μl
  2. Vortex gently, then centrifuge in a microcentrifuge for 5 seconds to bring the reaction mixture to the bottom of the tube.
  3. Incubate the reactions at 37°C for 60 minutes.
  4. Stop the reactions by placing the tubes in an ice bath for 5 minutes.
  5. Analyze the results of the reaction.
Click here for the manufacturer's maual

Materials:
  • 10X TE Buffer
  • 1M Tris HCl Buffer
  • Protein lysis buffer (PLB): 50 mM Tris (pH 7.5), 50 mM NaCl, dtt. sterilized by autoclaving
  • Start buffer: 20 mM Tris-HCl, pH 8.0 (At least 1 pH unit above the pI of substance)
  • Elution Buffer (20mM Tris-HCl, pH 8.0, 1 M NaCl).
  • Binding buffer: 4 M ammonium sulfate 132.14 g/mol in TE (Tris- EDTA)
  • Wash buffer: 1.3 M ammonium sulfate in TE
  • Equilibrating buffer: 2 M ammonium sulfate in TE
Procedures:
  1. Pick single colony of transformed C41 cells to 5ml LB solution with 1x antibiotics to grow starter.
  2. 1% Inoculation in two 1L conical flask, each with 250 ml 2XYT solution 1x antibiotics overnight.
  3. Spin down 100ml cells in 50 ml falcon. Discard the supernatant.
  4. Wash cell pellet with 40 ml cool TE buffer.
  5. Re-suspend cells with cold 15 ml Protein Lysis Buffer (PLB).
  6. Sonicate on ice.
  7. Spin at 4°C at 13000 rpm for 5 min
  8. Collect the supernatant and dialysis overnight.
  9. Pipette 900 µl of room temperature SOC or LB media into the mixture.
  10. Purify proteins with ion exchange column using start buffer (20 mM Tris-HCl, pH 8.0)
  11. Apply the sample at 5 ml/min for 5 ml columns by pumping it onto the column.
  12. Trace the proteins by naked eyes.
  13. Elute amajLime and mRFP at 0 µM and 30 µM NaCl respectively.
  14. Purify the proteins by HIC column followed by Ion exchange chromatography. Wash the column with 3 mL of Equilibrium buffer after HIC matrix resuspension.
  15. Remove the female luer for buffer running through the column to the waste collection tube.
  16. Mix 1 volume of protein sample to 1 volume of Binding Buffer (~200 µl).
  17. Transfer all 400 µl of protein/Binding Buffer mix to the column carefully without disturbing the top of the bed containing the settled HIC matrix.
  18. Trace the fluorescent protein by naked eyes or blue light.
  19. Wash the resin with 1 ml Wash buffer when the meniscus reaches the top of the bed and the run-through is collected in the waste collection tube.
  20. Add 1 ml TE buffer to the resin and the run-through without fluorescent proteins is collected in the waste collection tube.
  21. Continue adding TE buffer to run through fluorescent proteins and collect with Eppendorf.
  22. Run SDS-PAGE to determine purity.
  23. Determine protein concentration by refractometer.

  1. Prepare the buffers as follow:
    2. pH Composition
    2 0.2M KCl/HCl
    4 3M acetate buffer
    6 0.5M MES buffer
    7 1M PIPES buffer
    8 1M HEPES buffer
    10 0.2M NaHCO3/NaOH
    12 0.2M KCl/NaOH
  2. Diluted into buffers ranging in pH from 2-12 in 96-well plates.
  3. Determine absorbance/ fluorescence by Plate reader.

     

Email: igemcuhk1617@gmail.com