Team:HokkaidoU Japan/Protocols

=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)''' {|border="1" class="protocol" | | |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 ΔOD_550nm = 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 ==Bacterial Transformations== ==Mini-prep (Alkaline SDS Method)== ==PCR== ==Restriction Enzyme Digestions== ==DNA ligation== ==Agarose gel electrophoresis== ==Electroporation==