Team:NTU-LIHPAO-Taiwan/Protocols

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
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