Team:NTU-LIHPAO-Taiwan/Experiments

NTU-LIHPAO-Taiwan

Preparation of Competent Cells (E.coli)
Materials and Reagents :
  1. Escherichia coli DH5α
  2. LB broth (0.5% yeast extract, 1% tryptone, 1% NaCl)
  3. TB buffer (10 mM PIPES, 15 mM CaCl2-2H2O, 250 mM KCl, 55 mM MnCl2•4H2O)
  4. DMSO
  5. Liquid nitrogen
Equipment :
  1. Ice
  2. Shaker
  3. Spectrometer
  4. Centrifuge
Procedure :
  1. Inoculate 10 μL Escherichia coli DH5α into 10mL LB broth (1:1000)
  2. Grow for 12-16 hours at 37℃ with shaking
  3. Inoculate 500 μL Escherichia coli DH5α into 50mL LB broth (1:100)
  4. Grow for 2 hours at 37℃ with shaking to O.D.600=0.4-0.6
  5. Split the cell into two Falcon tubes, both contain 25 mL Escherichia coli DH5α
  6. Centrifuge at 4℃, 3000 rpm(800G) for 10 minutes
  7. Discard the supernatant
  8. Resuspend the cell pellet by gently adding 8.5mL TB buffer (1/3 V)
  9. Incubate in an ice bath for 10 minutes
  10. Centrifuge at 4℃, 3000 rpm(800G) for 10 minutes
  11. Discard the supernatant using pipetman
  12. Resuspend the cell pellet by gently adding 2 mL TB buffer (1/12.5 V)
  13. Add 150 μL DMSO (7%)
  14. Incubate in an ice bath for 10 minutes
  15. Transfer the cell to new eppendorfs with 60 μL per tube
  16. Freeze the cell in liquid nitrogen
  17. Store the cell at -80℃
DNA Dissolution
Materials and Reagents :
  1. DNA (BioBricks/synthesized)
  2. ddH2O
Procedure :
  1. Add 10 μL ddH2O
  2. Wait for 1 minute until the DNA dissolve
  3. Pipet several times and transfer the DNA to an eppendorf
Agarose Gel Electrophoresis
Materials and Reagents :
  1. Agarose
  2. 1X TAE (40 mM Tris-acetate, 1 mM EDTA)
  3. Tracking dye
  4. Marker (1kb/100bp)
  5. EtBr
  6. ddH2O
Equipment :
  1. Gel tray
  2. Well comb
  3. Electrophoresis tank
  4. UV detector
Procedure :
    Casting an Agarose Gel (m/v = 1.0% or 1.5%)
  1. Measure out appropriate mass of agarose powder into a serum bottle
  2. Add appropriate volume of 1X TAE
  3. Microwave until agarose is fully dissolved
  4. Let agarose solution cool down to acceptable temperature for bare hands
  5. Pour the solution into a gel tray with the well comb in place, and push the bubbles away with a pipette tip
  6. Let sit at room temperature for 30 minutes, until it has completely solidified
    7. Loading Samples and Running the Gel
  7. Place the gel as well as its tray into the electrophoresis tank containing 1X TAE
    8. (Make sure that the surface is higher than the top of the gel and not overflow)
  8. Mix the samples with tracking dye (1/10 V) sufficiently
  9. Load the samples into the each well
  10. Load marker (usually in the first and last lane)
  11. Set an appropriate voltage (full/half) and run the gel for 15-20 minutes
    12. Imaging the Gel
  12. Put the gel into a container filled with 1X TAE and EtBr, staining for 5 minutes
  13. Replace EtBr solution with water and destain for 3 minutes
  14. Put the gel in an UV detector and record the result
Gel Extraction
Materials and Reagents :
  1. Gel/PCR buffer
  2. Gel/PCR DNA Fragments Extraction Kit (Geneaid)
  3. W1 buffer
  4. Wash buffer
  5. DF Column & Collection tube
  6. ddH2O
Equipment :
  1. Cutter knife
  2. Eppendorf
  3. Vortex mixer
  4. Dry bath incubator
  5. Mini Centrifuge
  6. Microcentrifuge
Procedure :
    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 (<300μL)
  2. Transfer the gel slice to an eppendorf
  3. Add 500 μL Gel/PCR buffer to the sample and mix by vortex
  4. Incubate at 60℃ for 10-15 minutes to ensure the gel slice has been completely dissolved (invert the tube every 2-3 minutes)
  5. Cool the dissolved sample mixture to room temperature
    6. DNA Binding
  6. Place the DF Column in a 2 mL Collection tube
  7. Transfer 800 μL of the sample mixture to the DF Column
  8. Spin down for approximately 20 seconds
  9. Discard the flow-through and place the DF Column back in the Collection tube
    10. (If the sample mixture is more than 800μL, repeat the DNA binding step)
    Wash
  10. Add 400 μL W1 buffer into the DF Column
  11. Spin down for approximately 20 seconds
  12. Discard the flow-through and place the DF Column back in the Collection tube
  13. Add 600μL wash buffer (contains ethanol) into the DF Column
  14. Let stand for 1 minute at room temperature
  15. Spin down for approximately 30 seconds
  16. Discard the flow-through and place the DF Column back in the Collection tube
  17. Centrifuge at 12000 rpm for 3 minutes to dry the column matrix
    18. (Can be done twice)
  18. Discard the flow-through
    19. DNA Elution
  19. Transfer the dried DF Column to a new eppendorf (cap cut)
  20. Add 30μL of 37℃ ddH2O into the center of the column matrix
  21. Let stand for at least 2 minutes to ensure that ddH2O is completely absorbed
  22. Centrifuge at 12000 rpm for 2 minutes to elute the purified DNA
  23. Re-add the subnatant into the center of the column matrix
  24. Centrifuge at 12000 rpm for 2 minutes to elute the purified DNA
DNA Ligation
Materials and Reagents :
  1. DNA (insert & vector)
  2. Ligation high ver.2 (10% T4 DNA Ligase, 50 mM Tris (hydroxymethyl) aminomethane, 1% glycerol)
Equipment :
  1. Cooling dry bath incubator
Procedure :
  1. Table
    Components Volume (μL)
    Insert x
    Vector y
    Ligation High 1
    Total 7
            Molar ratio: insert/vector = 3/1
            x + y = 6
  2. Gently mix the solution by pipetting up and down
  3. Incubate
    1. at 16℃ for 2 hours, or
    2. at 37℃ for 1 hour, or
    3. at 4℃ overnight
  4. Proceed with bacterial transformation
Plasmid DNA Extraction Using Alkaline Lysis Method (E. coli)
Materials and Reagents :
  1. Escherichia coli DH5α (with plasmid)
  2. LB broth
  3. Resuspension solution (MPI, with RNAase)
  4. Lysis solution (MPII)
  5. Neutralizing solution (MPIII)
  6. Isopropanol
  7. 70% ethanol
  8. ddH2O
Equipment :
  1. Shaker
  2. Centrifuge
  3. Vortex mixer
Procedure :
  1. Grow bacteria in LB broth with appropriate antibiotics at 37℃ overnight with shaking
  2. Transfer 1.5mL culture to an eppendorf
  3. Centrifuge at 4℃, 5000rpm for 5 minutes
  4. Discard the supernatant
  5. Add 150 μL of resuspension solution (MPI, with RNAase) into each tube, pipet several times, and vortex to completely resuspend the cell pellet
  6. Add 150 μL of lysis solution (MPII), gently invert the tubes about 20 times, and then let the sample mixture stand at room temperature for 2 minutes
  7. Add 150 μL of neutralizing solution (MPIII) and mix by inverting the tubes about 20 times. Bacterial chromosomal DNA and proteins can be seen as white precipitates
  8. Centrifuge at 4℃, 15000 rpm for 15 minutes
  9. Carefully transfer 400 μL of the supernatant to a new eppendorf
    10. (Step 8 & 9 can be done twice)
  10. Add 400 μL (same volume as the supernatant) isopropanol, and shake up the tubes as vigorously as one can
  11. Centrifuge at 4℃, 15000 rpm for 15 minutes
  12. Discard the supernatant
  13. Add 200 μL of 70% ethanol to wash out the salts
  14. Centrifuge at 4℃, 15000 rpm for 5 minutes
  15. Discard the supernatant and remove the remains as much as possible with pipetman
  16. Air dry for about 30 minutes
  17. Resuspend the DNA pellet with 20μL ddH2O
Transformation (E. coli)
Materials and Reagents :
  1. Competent cell (Escherichia coli DH5α)
  2. LB plate (0.5% yeast extract, 1% tryptone, 1% NaCl, 1.5% agar) (Amp+/CP+)
Equipment :
  1. -80℃ refrigerator
  2. Ice
  3. 37℃ incubator
  4. Super optimal broth (SOB) (37℃)
  5. Shaker
Procedure :
  1. Take out the competent cells from -80℃ refrigerator
  2. Thaw the cells on ice for 5 minutes
  3. Add 1 μL plasmid DNA into the competent cells, and mix gently by pipetting
  4. On ice for 10 minutes
  5. Heat shock at 37℃ for 3 minutes
  6. On ice for 2 minutes
  7. Add 150 μL 37℃ SOB into the mixture
  8. Place the tube at 37℃ with shaking for 1 hour(Amp+)/4 hours(CP+) respectively
  9. Spread the cells onto the LB plates (Amp+/CP+)
  10. Incubate the plates at 37℃ with shaking for 12-16 hours
Protein Extraction
Materials and Reagents :
  1. Plasmid/ BL21 competent cell
  2. LB plate
  3. LB broth
  4. Lysis buffer (0.05 M NaH2PO4, 0.3 M NaCl, 0.01 M imidazole)
Equipment :
  1. Shaking incubator
  2. Erlenmeyer flask
  3. Spectrophotometer
  4. Centrifuge
  5. Sonicator
Procedure :
  1. Transform plamid into BL21 (see transformation)
  2. Pick up a single colony and incubate into 2 ml LB/CP medium, then agitate them in a shaking incubator at 37℃ overnight
  3. Pour the culture into 100 ml LB/CP medium, then agitate until it reach A600 of 0.6
  4. Pour the culture into 50 ml Falcon tube, and centrifuge at 8000 rpm for 10 min
  5. Store the supernate and the pellets in the centrifuge tubes in a -80℃ freezer
  6. Use 6 ml lysis buffer to resuspend the cell pellet and transfer into a 15 ml centrifuge tube
  7. Lyse the cell with sonication
  8. Centrifuge 15 ml Falcon tube at 9000 rpm for 10 min
  9. Store the supernate as coarse extract
SDS-PAGE
Materials and Reagents :
  1. Solution A (acrylamide/ bis-acrylamide (29:1) 30% solution)
  2. Solution B (Tris (Tris base, 1.5 M) 90.8 gm; TEMED 1.8 ml; pH = 8.8/ 500 ml dH2O)
  3. Solution C (Tris (Tris base, 0.5 M) 6.0 gm; TEMED 0.4 ml; pH = 6.7/ 100 ml dH2O)
  4. 10% SDS
  5. dH2O
  6. APS (ammonium persulfate 100 mg/ 1 mL dH2O)
  7. Running buffer (1M Tris 12.5 ml; glysine 7.7g/ 500 ml dH2O)
  8. Sample buffer(5X) (Tris 250 mM; EDTA2•Na, 10 mM; SDS(10%); glycerol (10%); brilliant blue; β-mercaptoethanol, 5% X5)
  9. Protein marker
Equipment :
  1. Gel Caster
  2. Accessories for each gel
  3.     (1) One glass plate
  4.     (2) One aluminum notched plate
  5.     (3) Two 0.75-mm thick spacers
  6.     (4) One 0.75-mm thick 10-well comb
  7.     (5) One well-locating decal
  8. Power supply
Procedure :
  1. Assemble one glass plate and one alumimum plate with two spacers for each gel module
  2. Prepare the SDS gel solution (see the table below). Two 0.75mm mini gels need approximately 10 ml separation gel solution and 5 ml stacking solution. Add APS solution at the final step
  3. Mix the separation gel solution, and add the solution into the gel module until reaching the designated level. Then add isopropanol on the top of the gel. It takes about 40 min to complete the polymerization step
  4. Remove the isopropanol by using a small strip of filter paper. Fill up the stacking gel solution until reaching the top. Insert comb into the gel solution as soon as possible. It takes 10 min to complete the polymerization step
  5. Remove the comb and set the gel in the cell buffer dam. Pour the running buffer into the inner chamber and keep pouring after overflow until the buffer surface reaches the required level in the outer chamber
  6. Use a micropipette to clean up every single well with the running buffer
  7. Mix protein samples with 1/5 volumes of 5X sample buffer. Heat the mixture at 100oC for 10 min. Let it cool down to room temperature
  8. Load protein sample and the protein marker into the well
  9. Run the electrophoresis at 70 V for 20 min, and then 150 V for 55 min
  10. Remove the two spacer and lift the glass plate, and remove the stacking gel and the remaining gel is ready for CBR staining and Western blot
(Unit: ml) Separation gel Stacking gel
Percentage 18% 20% 4%
Solution A 6 6.5 0.66
Solution B 2.5 2.5 -
Solution C - - 1.24
10% SDS 0.1 0.1 0.05
dH2O 1.35 0.85 2.95
APS(10%) 0.05 0.05 0.1
Total Volume 10 10 5
Western Blot
Materials and Reagents :
  1. Transfer buffer (25 mM Tris; 0.192 M glycine)
  2. Methanol
Equipment :
  1. Semi-dry trnasfer cell
  2. PVDF membrane
  3. 3 mm Filter paper
  4. Square box
  5. Rotary shaker
Procedure :
  1. Equilibrate the filter paper and SDS-PAGE gel by soaking them with transfer buffer
  2. Rinse the PVDF membrane with 100% methanol for a few second and equilibrate it with transfer buffer
  3. Place the elements in the following order onto the semi-dry transfer cell from bottom to top: filter paper, PVDF membrane, SDS-PAGE gel, filter paper
  4. Set up the current at 0.09 A and run for 75 min
Immunostaining
Materials and Reagents :
  1. Gelatin-NET (0.25% gelatin, 50 mM Tris, 0.15 M NaCl, 5 mM EDTA, 0.05% Tween-20)
  2. Nonfat-milk (3%/ TBST)
  3. Primary antibody (anti-Histag, mouse)
  4. Secondary antibody (anti-mouse IgG HRP, goat)
  5. TBST (0.01 mM Tris, 0.1% Tween-20, 0.15 M NaCl)
  6. HRP substrate (Luminata)
Equipment :
  1. Square box
  2. Rotary shaker
  3. UVP Biospectrum
Procedure :
  1. Put the membrane in the square box block the membrane with the 3% milk/TBST at room temperature for 1hr
  2. Discard the milk and replace it with primary antibody at 4℃ overnight
  3. Recycle the antibody solution. Wash the membrane with TBST three times, 5 min each
  4. Soak membrane into secondary antibody for 1 hr
  5. Discard the anti-mouse HRP solution and wash it with TBST three times, 5 min each
  6. Add HRP substrate on the membrane for 1 min
  7. Soak the membrane into ddH2O to remove the substrate
  8. Observe the result with UVP Biospectrum
Thawing Caco-2 cells
Materials and Reagents :
  1. 1 ml frozen Caco-2 cells in cryovial
  2. Cell culture medium (MEM/ 10% FBS/1% PSG)
  3. Trypan blue solution
  4. 10 cm culture dish
Equipment :
  1. Biological Safety Cabinet
  2. Centrifuge
  3. Hemocytometer
  4. Optical microscope
  5. Suction machine
Procedure :
  1. Pre-warm medium in 37˚C water bath
  2. Open a biological safety cabinet
  3. Clean materials, reagents and equipments before moving them into the hood
  4. Thawing Caco-2 cells in 37˚C water bath and transfer the cells into a 15 ml Falcon tube
  5. Add 9 ml cell culture medium very slowly
  6. Centrifuge at 1000 rpm for 5 min
  7. Decant the supernate and resuspend cells with 10 ml cell culture medium
  8. Mix 10 μl cell suspension and 10 μl trypan blue solution and count total cell number with a hemocytometer
  9. Add all cell suspension to a 10 cm culture dish
  10. Put the dish into incubator
Freezing Caco-2 cells
Materials and Reagents :
  1. Caco-2 cell in 10 cm culture dish
  2. Cell culture medium
  3. PBS (phosphate buffer saline) (10 mM NaH2PO3, 0.15 M NaCl)
  4. Trypsin (0.5%, 1 mM EDTA)
  5. Trypan blue solution
  6. Cryovials
  7. Cell freezing media (50% FBS, 40% cell culture medium, 10% DMSO)
Equipment :
  1. Biological Safety Cabinet
  2. Centrifuge
  3. 10 cm culture dish
  4. Hemocytometer
  5. Optical microscope
  6. Suction machine
  7. Freezing apparatus
  8. -80˚C refrigerator
  9. Liquid nitrogen tank
Procedure :
  1. Pre-warm medium in 37˚C water bath
  2. Open a biological safety cabinet
  3. Clean materials, reagents and equipments before moving them into the hood
  4. Take a 10 cm culture dish with Caco-2 cell
  5. Discard the medium with a suction machine
  6. Wash the cells with 5 ml PBS
  7. Add 1 ml 0.5% trypsin and incubate at 37˚C for 7 minutes
  8. Wash out all the cells from the surface of the dish by pipetting the fresh complete culture medium (9 ml) all over the surface
  9. Transfer the cell suspension to a 15 ml centrifuge tube
  10. Mix 10 μl cell suspension and 10 ul trypan blue solution and count total cell number with a hemocytometer
  11. Add about 1x106 cells per cryovial, and add cell freezing media
  12. Freeze the cryovials in a controlled rate freezing apparatus, decreasing the temperature approximately 1°C per minute in a -80°C refrigerator for more than 3 hour, then move them into liquid nitrogen tank
Caco2 Cell passage
Materials and Reagents :
  1. Caco-2 cell in 10 cm dish
  2. Cell culture medium
  3. PBS
  4. Trypsin
  5. Trypan blue solution
  6. 10 cm culture dish
Equipment :
  1. Biological Safety Cabinet
  2. Centrifuge
  3. Hemocytometer
  4. Optical microscope
  5. Suction machine
Procedure :
  1. Pre-warm medium in 37˚C water bath
  2. Open a biological safety cabinet
  3. Clean materials, reagents and equipment before moving them into the cabinet
  4. Take a 10 cm dish with Caco-2 cell
  5. Discard the medium with a suction machine
  6. Wash the cells with 5 ml PBS
  7. Add 1 ml 0.5% trypsin and incubate at 37˚C for 7 minutes
  8. Wash out all the cells from the surface of the dish by pipetting the fresh complete culture medium (9 ml) all over the surface
  9. Transfer the cell suspension to a 15 ml Falcon tube
  10. Mix 10 μl cell suspension and 10 μl trypan blue solution and count total cell number with a hemocytometer
  11. Transfer appropriate amounts of cell suspension to new dishes (1/4 X)
  12. Put the dish into incubator
Seeding in transwell
Materials and Reagents :
  1. Caco-2 cell in 10 cm dish
  2. Cell culture medium
  3. 1X PBS (phosphate buffer saline)
  4. Trypsin
  5. Trypan blue solution
Equipment :
  1. Biological Safety Cabinet
  2. Centrifuge
  3. Hemocytometer
  4. Optical microscope
  5. Suction machine
  6. 6-well plate
  7. Millicell plate insert (PIHP03050)
Procedure :
  1. Open 6-well plate, add 1.5 mL medium to each large well (6 wells) in which we select to put inserts
  2. Add 2 ml PBS/well to other wells
  3. Put insert in selected well
  4. Take a 10 cm dish with Caco-2 cell
  5. Discard the medium with a suction machine
  6. Wash the cells with 5 ml PBS
  7. Add 1 ml trypsin and incubate at 37˚C for 7 minutes
  8. Wash out all the cells from the surface of the dish by pipetting the fresh complete culture medium (9 ml) all over the surface
  9. Transfer the cell suspension to a 15 ml centrifuge tube
  10. Mix 10 μl cell suspension and 10 μl trypan blue solution and count total cell number with a hemocytometer
  11. Cell count: 70/ 2 * orginal volume *100 (*104)= 35 X 104 cell/mL
  12. Seeding 42 x 104 cell/well
  13. Put the dish into incubator
Measure TEER
Materials and Reagents :
  1. Caco-2 cells seeded in transwell
Equipment :
  1. Biological Safety Cabinet
  2. Millicell ERS
Procedure :
  1. Prepare a biological safety cabinet for experiment
  2. To sterilize electrode of Millicell ERS, soak electrode in 70% alcohol, and air dry for 15 sec, soak it in cell culture medium for another 15 min
  3. Measure TEER value of each well (n=3)
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