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From 2010.igem.org

Contents 1 2010/08/26(Bambi75) 2 Make Plates 2.1 member 2.2 Materials 2.3 Procedure 3 Separation of A.xylinum 3.1 member 3.2 Materials 3.3 Procedure 4 PCR 4.1 member 4.2 Materials 4.3 Procedure 5 PCR 5.1 member 5.2 Materials 5.3 Procedure

2010/08/26(Bambi75)

Make Plates

member

NEX , Bambi75 and watachin

Materials

• RO water 200ml
• LB Broth 4g
• Cam(50μg/L) 10ml

Procedure

① mix materials.

② Divide ①equally and make 10 plates.

Separation of A.xylinum

member

easily and naoto

Materials

• the plate(made on August 25th).

Procedure

①extract material 2ml.

②centrifuge ① 10000rpm/5min.

PCR

member

same above

Materials

• 2×PCR buffer 25×4μl
• 2mM dNTP 10×4μl
• 10mM primer(sense)bcsA,B,C and D 2.5μl each
• 10mM primer(antisense)bcsA,B,C and D 2.5μl each
• template DNA a little
• Q water 9×4μl
• KOD FX 0.5×4μl

Procedure

①mix all materials for 4 tubes.

②elongation

• bcsA,bcsB and bcsC
  • 94℃ 2min
  • 98℃ 10sec☆
  • 55℃ 30sec
  • 68℃ 4min★
  • 68℃ 7min
  • 10℃　∞
• bcsD
  • 94℃ 2min
  • 98℃ 10sec☆
  • 55℃ 30sec
  • 68℃ 1min★
  • 68℃ 7min
  • 10℃　∞

※30cycle ☆ to ★.

PCR

member

NEX , Bambi75 and watachin

Materials

• sterilized water 71μl
• Ex taq buffer 10μl
• dNTP mix 8μl
• Ex taq 1μl
• primer(bcsC sense/antisense) 5μl each

Procedure

①mix materials

②divide ① into 2 tubes.

③elongation

• • 95℃ 3min
  • 96℃ 1min☆
  • 55℃ 7min
  • 72℃ 1min★
  • 10℃ ∞

※30 cycle ☆to★.