Team:NTU-LIHPAO-Taiwan/Notebook

NTU-LIHPAO-Taiwan

Preparation of Competent Cells (E.coli)
Materials and Reagents :
  1. Escherichia coli DH5α
  2. LB broth
  3. TB buffer
  4. DMSO
  5. Liquid nitrogen
Equipment :
  1. Ice
  2. Shaker
  3. Spectrometer
  4. Centrifuge
Procedure :
  1. Inoculate 10μL Escherichia coli DH5α into 10mL LB broth (1:1000)
  2. Grow for 12-16 hours at 37℃ with shaking
  3. Inoculate 500μL 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℃, 3000rpm(800G) for 10 minutes
  7. Discard the supernatant
  8. Resuspend the cell pellet by gently adding 8.5mL TB buffer (1/3 V)
  9. On ice for 10 minutes
  10. Centrifuge at 4℃, 3000rpm(800G) for 10 minutes
  11. Discard the supernatant using pipetman
  12. Resuspend the cell pellet by gently adding 2mL TB buffer (1/12.5 V)
  13. Add 150μL DMSO (7%)
  14. On ice for 10 minutes
  15. Transfer the cell to new eppendorfs with 60μL 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 10μL 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
  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. Let agarose solution cool down to acceptable temperature for bare hands
  4. Pour the solution into a gel tray with the well comb in place, and push the bubbles away with a pipette tip
  5. Let sit at room temperature for 30 minutes, until it has completely solidified
    6. Loading Samples and Running the Gel
  6. Place the gel as well as its tray into the electrophoresis tank containing 1X TAE
    7. (Make sure that the surface is higher than the top of the gel and not overflow)
  7. Mix the samples with tracking dye (1/10 V) sufficiently
  8. Load the samples into the each well
  9. Load marker (usually in the first and last lane)
  10. Set an appropriate voltage (full/half) and run the gel for 15-20 minutes
    11. Imaging the Gel
  11. Put the gel into a container filled with 1X TAE and EtBr, staining for 5 minutes
  12. Replace EtBr solution with water and destain for 3 minutes
  13. Put the gel in an UV detector and record the result
Gel Extraction
Materials and Reagents :
  1. Gel/PCR buffer
  2. W1 buffer
  3. Wash buffer
  4. ddH2O
Equipment :
  1. Cutter knife
  2. Eppendorf
  3. Vortex mixer
  4. Dry bath incubator
  5. DF Column & Collection tube
  6. Mini Centrifuge
  7. 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 500μL 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 2mL Collection tube
  7. Transfer 800μL of the sample mixture to the DF Column
  8. Discard the flow-through and place the DF Column back in the Collection tube
    9. (If the sample mixture is more than 800μL, repeat the DNA binding step)
    Wash
  9. Add 400μL W1 buffer into the DF Column
  10. Spin down for approximately 20 seconds
  11. Discard the flow-through and place the DF Column back in the Collection tube
  12. Add 600μL wash buffer (contains ethanol) into the DF Column
  13. Let stand for 1 minute at room temperature
  14. Spin down for approximately 30 seconds
  15. Discard the flow-through and place the DF Column back in the Collection tube
  16. Centrifuge at 12000 rpm for 3 minutes to dry the column matrix
    17. (Can be done twice)
  17. Discard the flow-through
    18. DNA Elution
  18. Transfer the dried DF Column to a new eppendorf (cap cut)
  19. Add 30μL of 37℃ ddH2O into the center of the column matrix
  20. Let stand for at least 2 minutes to ensure that ddH2O is completely absorbed
  21. Centrifuge at 12000 rpm for 2 minutes to elute the purified DNA
  22. Re-add the subnatant into the center of the column matrix
  23. 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
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 150μL of resuspension solution (MPI, with RNAase) into each tube, pipet several times, and vortex to completely resuspend the cell pellet
  6. Add 150μL 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 150μL 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℃, 15000rpm for 15 minutes
  9. Carefully transfer 400μL of the supernatant to a new eppendorf
    10. (Step 8&9 can be done twice)
  10. Add 400μL (same volume as the supernatant) isopropanol, and shake up the tubes as vigorously as one can
  11. Centrifuge at 4℃, 15000rpm for 15 minutes
  12. Discard the supernatant
  13. Add 200μL of 70% ethanol to wash out the salts
  14. Centrifuge at 4℃, 15000rpm 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 (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 1μL 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 150μL 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
Equipment :
  1. 15/ 50ml conial tube
  2. Shaking incubator
  3. Erlenmeyer flask
  4. Spectrophotometer
  5. Centrifuge
  6. Sonicator
  7. Eppendorf
Procedure :
  1. Transform plamid into BL21 (see transformation)
  2. Pick up a single colony and incubate into 2ml LB/CP medium, then agitate them in a shaking incubator at 37℃ overnight
  3. Pour the culture into 100ml LB/CP medium, then agitate until it reach A600 of 0.6
  4. Pour the culture into 50ml conical tube, and centrifuge at 8000rpm for 10 min
  5. Store the supernate and the pellets in the centrifuge tubes in a -80℃ freezer
  6. Use 6ml lysis buffer(NP-10) to resuspend the cell pellet and transfer into a 15ml centrifuge tube
  7. Lyse the cell with sonication
  8. Centrifuge 15ml conial tube at 9000rpm 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/ 500ml)
  3. Solution C (Tris (Tris base, 0.5 M) 6.0 gm; TEMED 0.4 ml; pH = 6.7/ 100ml)
  4. 10% SDS (1kb/100bp)
  5. ddH2O
  6. APS
  7. Running buffer (1M Tris 12.5 ml; glysine 7.7g/ 500ml)
  8. Sample buffer(5X) (Tris base, 125mM X5; EDTA2•Na, 2mM X 5; SDS(2% X2); 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 10ml separation gel solution and 5ml 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 70V for 20 min, and then 150V 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 5% 7.5% 10% 12.5% 15% 20% 4%
Solution A 16.5 2.5 3.35 4.15 5 6.5 0.66
Solution B 2.5 2.5 2.5 2.5 2.5 2.5 -
Solution C - - - - - - 1.24
10% SDS 0.1 0.1 0.1 0.1 0.1 0.1 0.05
dH2O 5.7 4.85 4 3.2 2.35 0.85 2.95
APS(10%) 0.05 0.05 0.05 0.05 0.05 0.05 0.1
Total Volume 10 10 10 10 10 10 5
Western Blot
Materials and Reagents :
  1. Transfer buffer ( Tris 25mM; glycine 0.192M)
  2. Methanol
  3. 10 X TBST (1M Tris pH =8.0; Tween 20, 50ml; NaCl 43.5g)
Equipment :
  1. Semi-dry trnasfer cell
  2. PVDF membrane
  3. Nitrocellulose paper
  4. Square Petri dish
  5. Rotary shaker
Procedure :
  1. Equilibrate the nitrocellulose 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: nitrocellulose paper, PVDF membrane, SDS-PAGE gel, nitrocellulose paper
  4. Set up the current at 0.09A and run for 75 min
Immunostaining
Materials and Reagents :
  1. 3% milk
  2. Antibody solution
  3. Secondary antibody (anti-mouse HRP)
  4. TBST
  5. Western HRP substrate
Equipment :
  1. Square petri dish
  2. Rotary shaker
  3. UVP Biospectrum
Procedure :
  1. Put the membrane in the square petri dish block the membrane with the 3% milk/TBST at room temperature for 1hr
  2. Discard the milk and replace it with antibody solution(mouse His-probe Antibody) at 4℃ overnight
  3. Recycle the antibody solution. Wash the membrane with TBST three times, 5 min each
  4. Soak membrane into anti-mouse HRP solution 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 (minimum essential medium) (GIBCOTM #41500-034)/2.2g NaHCO3/ 0.11 g sodium pyruvate/20% FBS/1% PSA)
  3. 70% ethanol
  4. Trypan blue solution
Equipment :
  1. Laminar flow hood
  2. Auto pipette
  3. Centrifuge
  4. 15 ml centrifuge tubes
  5. 10 cm dish
  6. Hemocytometer
  7. Optical microscope
  8. Suction machine
Procedure :
  1. Pre-warm medium in 37˚C water bath
  2. Open a laminar flow hood
  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 15ml centrifuge 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 ul 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 dish
  10. Write date, cell line and passage number
  11. Put the dish into incubator
Freezing Caco-2 cells
Materials and Reagents :
  1. Caco-2 cell in 10 cm dish
  2. Cell culture medium (MEM (minimum essential medium) (GIBCOTM #10370-021)/10% FBS/ 2 mM glutamine
  3. 1X PBS (phosphate buffer saline)
  4. 0.5% trypsin
  5. Trypan blue solution
  6. 70% ethanol
  7. Cell freezing media (50% FBS, 40% cell culture medium, 10% DMSO)
Equipment :
  1. Laminar flow hood
  2. Auto pipette
  3. Centrifuge
  4. 15 ml centrifuge tubes
  5. 10 cm dish
  6. Hemocytometer
  7. Optical microscope
  8. Suction machine
  9. Cryovials
  10. Freezing apparatus
  11. -80˚C refrigerator
  12. Liquid nitrogen tank
Procedure :
  1. Pre-warm medium in 37˚C water bath
  2. Open a laminar flow hood
  3. Clean materials, reagents and equipments before moving them into the hood
  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 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 (MEM (minimum essential medium) (GIBCOTM #10370-021)/10% FBS/ 2 mM glutamine
  3. 1X PBS (phosphate buffer saline)
  4. 0.5% trypsin
  5. Trypan blue solution
  6. 70% ethanol
Equipment :
  1. Laminar flow hood
  2. Auto pipette
  3. Centrifuge
  4. 15 ml centrifuge tubes
  5. 10 cm dish
  6. Hemocytometer
  7. Optical microscope
  8. Suction machine
Procedure :
  1. Pre-warm medium in 37˚C water bath
  2. Open a laminar flow hood
  3. Clean materials, reagents and equipments before moving them into the hood
  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 centrifuge tube
  10. Mix 10 ul 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. Write date, cell line and passage number
  13. Put the dish into incubator
Seeding in transwell
Materials and Reagents :
  1. Caco-2 cell in 10 cm dish
  2. Cell culture medium (MEM (minimum essential medium) (GIBCOTM #41500-034)/2.2 g NaHCO3/ 0.11 g sodium pyruvate/20% FBS/1% PSA
  3. 1X PBS (phosphate buffer saline)
  4. 0.5% trypsin
  5. Trypan blue solution
  6. 70% ethanol
Equipment :
  1. Laminar flow hood
  2. Auto pipette
  3. Centrifuge
  4. 15 ml centrifuge tubes
  5. 10 cm dish
  6. Hemocytometer
  7. Optical microscope
  8. Suction machine
  9. 6-well plate
  10. Millicell plate insert (PIHP03050)
Procedure :
  1. Open 6-well plate, add 1.5 mL medium to each large well (6 wells) which are going to be used
  2. Add 2 ml PBS/well to other wells
  3. Put transwell in larger 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 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 ul 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 (Seeding 42 x 104 cell/well)
  12. Write date, cell line and passage number
  13. Put the dish into incubator
Measure TEER
Materials and Reagents :
  1. Caco-2 cells seeded in transwell
  2. 70% ethanol
Equipment :
  1. Laminar flow hood
  2. Millicell ERS
Procedure :
  1. Prepare laminar hood for experiment
  2. To sterilize electrode of Millicell ERS, soak electrode in 70% alcohol for 15 min, and air dry for 15 sec
  3. Measure TEER value of each well (n=3)
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