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

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{{:Team:Tokyo_Metropolitan/Header}}
{{:Team:Tokyo_Metropolitan/Header}}
 +
==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★.

Revision as of 06:06, 8 September 2010


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★.

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