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Team:HokkaidoU Japan/Protocols
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* Preparation of Competent cells (''E. coli'' DH5a) | * Preparation of Competent cells (''E. coli'' DH5a) | ||
* Bacterial Transformations | * Bacterial Transformations | ||
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* Agarose gel electrophoresis | * Agarose gel electrophoresis | ||
* Electroporation | * Electroporation | ||
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Revision as of 16:20, 21 September 2010
since 13 Jul 2010
since 1 Aug 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
- Single colony isolation on LB plate
- incubate the plate for 15-19 hrs at 37C
- lift a colony into 2 mL of LB
- culture cells at 37C for 12-16 hrs at 180-200 rpm
- transfer 30 uL, 100 uL, 300 uL of the culture into 100 mL SOB medium, respectively
- culture cells at 20C (for 24 hrs over) at 180-200 rpm (to ΔOD550nm = 0.5~0.6)
- leave the 300 mL flask for 10 min on ice
- transfer the culture into two 50 mL Falcon tube
- centrifuge 7500 rpm at 4C for 20 min (TOMY TA-22 rotor), and discard sup
- suspend the pellet in ice-cold 15 mL of TB (Transformation Buffer)(7.5 mL/tube)
- centrifuge 7500 rpm at 4C for 2 min (TOMY TA-22 rotor), and discard sup
- suspend the pellet in ice-cold 3.2 mL of TB
- add 0.24 mL of DMSO (stirring, bit by bit)
- leave the 50 mL Falcon tube for 10 min on ice
- dispense 50 uL into 0.5 mL tube
- freeze the suspension in liquid nitrogen
- store at -80C
