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Team:Tokyo Metropolitan/Notebook/Fiber/2010/08/26
From 2010.igem.org
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★.
