Team:Tokyo Metropolitan/Notebook/Fiber/2010/08/12

E.coli Fiber Project Notebook

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 and divide 1) into 3 tips.

2.*mix K12bcsAsense , K12bcsAantisense and 2)

mix K12bcsBsense , K12bcsBantisense and 2)

mix K12bcsCsense , K12bcsCantisense and 2)

3.dip E.coliK12strain into 3).

4.setting tips and elongation in thermal cycler

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