Team:HokkaidoU Japan/Protocols

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Revision as of 16:22, 21 September 2010

Protocols

  • Contents

    • Preparation of Competent cells (E. coli DH5a)
    • Bacterial Transformations
    • Mini-prep (Alkaline SDS Method)
    • PCR
    • Restriction Enzyme Digestions
    • DNA ligation
    • Agarose gel electrophoresis
    • Electroporation

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

filtration (0.2 um), store at 4C

Method

  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

Mini-prep (Alkaline SDS Method)

PCR

Restriction Enzyme Digestions

DNA ligation

Agarose gel electrophoresis

==Electroporation==
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