Team:Tokyo Metropolitan/Notebook/Fiber/2010/08/26

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Contents

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★.