Team:HokkaidoU Japan/Protocols

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Protocols

  • Preparation of Competent cells (E. coli DH5a)

    Reagents

    TB (Transformation Buffer)(at 4C, filtration)

    Final concentration
    1 M CaCl2 (at RT, autoclaved) 0.75 mL 15 mM
    4 M KCl (at RT, autoclaved) 3.125 mL 250 mM
    1 M MnCl2 (at 4C, autoclaved) 2.75 mL 55 mM
    1 M PIPES (pH 6.7 by NaOH, at 4C, filtration) 0.5 mL 10 mM
    Total 50 mL

    Procedure

    1. Single colony isolation on LB plate
    2. incubate the plate for 15-19 hrs at 37C
    3. lift a colony into 2 mL of LB
    4. culture cells at 37C for 12-16 hrs at 180-200 rpm
    5. transfer 30 uL, 100 uL, 300 uL of the culture into 100 mL SOB medium, respectively
    6. culture cells at 20C (for 24 hrs over) at 180-200 rpm (to ΔOD550nm = 0.5~0.6)
    7. leave the 300 mL flask for 10 min on ice
    8. transfer the culture into two 50 mL Falcon tube
    9. centrifuge 7500 rpm at 4C for 20 min (TOMY TA-22 rotor), and discard sup
    10. suspend the pellet in ice-cold 15 mL of TB (Transformation Buffer)(7.5 mL/tube)
    11. centrifuge 7500 rpm at 4C for 2 min (TOMY TA-22 rotor), and discard sup
    12. suspend the pellet in ice-cold 3.2 mL of TB
    13. add 0.24 mL of DMSO (stirring, bit by bit)
    14. leave the 50 mL Falcon tube for 10 min on ice
    15. dispense 50 uL into 0.5 mL tube
    16. freeze the suspension in liquid nitrogen
    17. store at -80C

  • Bacterial Transformations

    1. add DNA solution to thawed competent cells
    2. incubate the cells on ice for 30 min
    3. heat shock the cells by immersion in a pre-heated water bath at 42C for 60 sec
    4. incubate the cells on ice for 5 min
    5. add 200 uL of SOB broth
    6. incubate the cells at 37C for 2 hrs while the tubes are shaking
    7. plate 200 uL of the transformation onto the dish
    8. incubate the plate at 37C for 12-14 hrs

  • Mini-prep (Alkaline SDS Method)

    Reagents

    Solution I (at RT, filtration 0.2 um, 50 mL)

    Final concentration
    Glucose (at RT) 0.45 g 50 mM
    1 M Tris-HCl (pH8.0, at RT, autoclaved) 1.25 mL 25 mM
    0.5 M EDTA (pH8.0, at RT, autoclaved) 1 mL 10 mM
    Total 50 mL


    Solution II (at RT, filtration 0.2 um, 20 mL)

    Final concentration
    10 N NaOH (at RT) 0.4 mL 0.2 N
    10% SDS (at RT, filtration) 2 mL 1%
    Total 20 mL


    Solution III (at RT, filtration 0.2 um, 50 mL)

    Final concentration
    5 M CH3COOK 30 mL 3 M
    CH3COOH 5.75 mL
    H2O 14.25 mL
    Total 50 mL

    Procedure

    1. lift colony E. coli into 2 mL LB contained antibiotics
    2. culture cells at 37C for 16-20 hrs at 180-200 rpm
    3. transfer 1.2-1.5 mL of culture into 1.5 mL tube
    4. centrifuge the culture at 15,000 rpm for 1 min at 4C and discard sup
    5. suspend the pellet in ice-cold 100 uL of Solution I
    6. add 200 uL of Solution II to the suspension
    7. mix by inverting the tube 10-20 times
    8. add ice-cold 150 uL of Solution III to the suspension
    9. mix by inverting the tube 10-20 times
    10. leave the tube for 5 min on ice
    11. add 10 uL of Chloroform
    12. mix by inverting the tube 5-10 times
    13. centrifuge the suspension at 15,000 rpm for 5 min at 4C
    14. transfer the supernatant into new 1.5 mL tube↓
    15. add equal volume of isopropanol and mix by voltexing
    16. leave the tube for 5 min at RT
    17. centrifuge the suspension at 15,000 rpm for 10 min at 4C and discard sup
    18. rinse the ppt by 70% EtOH and mix by voltexing
    19. centrifuge the suspension at 15,000 rpm for 2 min at 4C and discard sup
    20. dry up the ppt
    21. dissolve the ppt in 50 uL of TE (pH 8.0)
    22. add 1 uL of 10 mg/mL RNase A (4C and stock at –20C)
    23. incubate for 30 min at 37C
    24. PCIAA and CIAA extraction
    25. Ethanol precipitation
    26. dry up the ppt
    27. dissolve the ppt in 50 uL of TE (pH 8.0)

  • PCR

    Vector

    Standard reaction setup

    Component Volume
    10x PCR Buffer 5 uL
    2mM dNTPs 5 uL
    25mM MgSO4 3 uL
    Suffix-F primer 1 uL
    Prefix-R primer 1 uL
    Template DNA 1 uL
    KOD -Plus- Neo 1 uL
    DW X uL
    Total 50 uL

    Cycling conditions (2-step cycle)

    Predenature 94C 2 min
    Denature 98C 10 sec
    Extension 68C X sec (30 sec/kb)
    Hold 4C
    • 30-40 cycles



    Insert

    Standard reaction setup

    Component Volume
    10x PCR Buffer 5 uL
    2mM dNTPs 5 uL
    25mM MgSO4 3 uL
    EX-F primer 1 uL
    PS-R primer 1 uL
    Template DNA 1 uL
    KOD -Plus- Neo 1 uL
    DW X uL
    Total 50 uL

    Cycling conditions (2-step cycle)

    Predenature 94C 2 min
    Denature 98C 10 sec
    Extension 68C X sec (30 sec/kb)
    Hold 4C
    • 30-40 cycles

    Colony PCR

    • resuspend a colony into 10 uL of DW (template suspension)

    Standard reaction setup

    Component Volume
    template suspension 4.8 uL
    Quick Taq 5 uL
    Forward primer 0.1 uL
    Reverse primer 0.1 uL
    Total 10 uL

    Cycling conditions (2-step cycle)

    Predenature 94C 2 min
    Denature 94C 10 sec
    Extension 68C X sec (60 sec/kb)
    Hold 4C
    • 30-40 cycles

  • Restriction Enzyme Digestions

    c

  • DNA ligation

    d

  • Agarose gel electrophoresis

    DNA Weight Markers

  • Electroporation

    Preparation of electro-competent cells

    1. cell culture in 400 mL of SOB or LB and grow to ΔOD600 = 0.5~0.6
    2. dispense the medium into 8 Falcon 50 mL tube
    3. centrifuge at 3500 rpm for 5 min at 4C and discard sup
    4. add 5 mL of DW and suspend the ppt, mix 8 suspensions into single Falcon tube
    5. centrifuge at 3500 rpm for 5 min at 4C and discard sup
    6. add 40 mL of DW and suspend the ppt
    7. centrifuge at 3500 rpm for 5 min at 4C and discard sup
    8. add 10 mL of 10% Glycerol and suspend the ppt
    9. centrifuge at 3500 rpm for 5 min at 4C and discard sup
    10. add 10 mL of 10% Glycerol and suspend the ppt
    11. centrifuge at 3500 rpm for 5 min at 4C and discard sup
    12. add 5 mL of 10% Glycerol and suspend the ppt
    13. dispense 100 uL of the suspensions into 0.5 mL Eppendorf tube, respectively
    14. store at -80C freezer


    Electroporation


  • Ethanol presipitation

    1. add 1/10 volume of 3M CH3COONa
    2. add 2.5 volume of 100% ethanol (EtOH)
    3. incubate on ice for few min (longer incubation time and lower incubation temperature are critical for small fragments)
    4. centrifuge at 15,000 rpm for 10 min at 4C and discard sup
    5. wash precipitation with 100 uL of 70% EtOH (EtOH has to be cold)
    6. centrifuge at 15,000 rpm for 5 min at 4C and discard sup
    7. dry up the ppt (no EtOH should be left)
    8. resuspend ppt in wanted volume of TE

  • PCIAA and CIAA extraction

    h
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