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2010/08/12 Tuesday (Bambi75)

Experiment:PCR

member

NEX , bambi75 and watachin

Materials

• sterilized water 106.5μl
• Ex taq buffer 15μl
• dNTP 12μl
• Ex taq 1.5μl
• K12bcsA sense(10μmol/l)2.5μl
• K12bcsA antisense(10μmol/l)2.5μl
• K12bcsB sense(10μmol/l)2.5μl
• K12bcsB antisense(10μmol/l)2.5μl
• K12bcsC sense(10μmol/l)2.5μl
• K12bcsC antisense(10μmol/l)2.5μl
• E.coliK12strain

Equipment

• thermal cycler
• vortex mixer
• PCR-tubes
• pipette tip

Procedure

1)mix materials : sterilized water , Ex taq buffer , dNTP and Ex taq.

2)divide 1) into 3 tips.

3)mix K12bcsAsense , K12bcsAantisense and 2).

　mix K12bcsBsense , K12bcsBantisense and 2).

　mix K12bcsCsense , K12bcsCantisense and 2).

4)dip E.coliK12strain into 3).

5)setting tips and elongation in thermal cycler

• initialization 95℃ 3min
• denaturation 96℃1min
• annealing 55℃ 5min
• elongation 72℃ 1min (30 cycles from 95℃ to 72℃)
• reaction stop 10℃