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From 2010.igem.org

Contents 1 2010/09/06 Monday(NEX) 1.1 Experiment:subculture of E.coli 1.2 Experiment:electrophoresis 1.3 Experiment:Making LB plate 1.4 Experiment:Direct PCR 1.5 Experiment

2010/09/06 Monday(NEX)

Experiment:subculture of E.coli

Member
naoto, watachin, bambi75 and NEX

Material

• E.coli K12

Procedure

1. Pick up a culture of E.coli K12 and streak it to new culture(LB plate)

Experiment:electrophoresis

Member
Same above

Material

• PCR production (bcsA and bcsB from E.coli)
• 1×TAE buffer
• Agarose gel

Procedure
Follow to protocol4

Consequence
Each bands were not appeared

Experiment:Making LB plate

Member
naoto

Material

• Distilled water 200ml
• LB Broth 4g

Experiment:Direct PCR

Member
naoto, watachin, bambi75 and NEX

Materials

• sterilized water 213μl
• Ex taq buffer 30μl
• dNTP 24μl
• Ex taq 3μl
• K12bcsA sense(10μmol/l)5μl
• K12bcsA antisense(10μmol/l)5μl
• K12bcsB sense(10μmol/l)5μl
• K12bcsB antisense(10μmol/l)5μl
• K12bcsC sense(10μmol/l)5μl
• K12bcsC antisense(10μmol/l)5μl
• E.coli K12 strain

Procedure
Follow to protocol3 Direct PCR

Experiment

Member
Same above

Materials

• pSB1A3(25ng/μl) 1μl
• Competent cell JM109 50μl
• LB + chloramphenicol

Procedure

1. Mix pSB1A3 and competent cell
2. On ice (30min)
3. Heat shock 42℃ 45sec
4. On ice (2min)
5. Inoculate these onto LB plates
6. Incubate plates at 37℃

Consequence
Failure