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 ΔOD_550nm = 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

  File:Image:HokkaidoU Pictures DNA Marker.png

  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

  g

• PCIAA and CIAA extraction

  h