Team:Tokyo Metropolitan/Notebook/Fiber/2010/10/24

From 2010.igem.org

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naoto and watachin
naoto and watachin
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===Experiment:Colony PCR===
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===Preparation:making preculture for Miniprep ===
'''material'''
'''material'''
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*colony of E.coli
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*colony of ''E.coli''
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**bcsB:1~32
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**bcsA→No.21,22
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**bcsC:33~64
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**bcsB→No.10
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**bcsD:65~96
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**bcsC→No.54,64
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*other materials were same as <html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80PCR">protocol3</a></html>
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**bcsD→No.75
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*LB+Cam broth
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'''result'''
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'''procedure'''
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From the length of bands
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our procedure was largely same as <html><a href="https://2010.igem.org/Team:Tokyo_Metropolitan/Project/Fiber/Protocol#.E3.80.80Grow up culture of E.coli">protocol2</a></html>
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bcsC→No.54 and 64
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bcsD→No.75
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seem to be correct inserts
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===Experiment:Ligation===
===Experiment:Ligation===

Revision as of 19:45, 24 October 2010


E.coli Fiber Project Notebook

August 2010
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September 2010
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October 2010
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2010/10/24 Sunday (Naoto)

member

naoto and watachin

Preparation:making preculture for Miniprep

material

  • colony of E.coli
    • bcsA→No.21,22
    • bcsB→No.10
    • bcsC→No.54,64
    • bcsD→No.75
  • LB+Cam broth

procedure

our procedure was largely same as protocol2

Experiment:Ligation

material

see protocol8

  • vector
    • pUC19(digested by XbaI)
  • Insert
    • bcsA(digested by XbaI+SpeI)
    • bcsB(digested by XbaI+SpeI)
    • bcsC(digested by XbaI+SpeI)
    • bcsD(digested by XbaI+SpeI)

procedure

refer to　protocol8

Experiment:Transformation

material

see protocol9

  • Ecos JM109/Nova Blue(Competent Cell)

procedure

refer to　protocol9

Experiment:Miniprep

material

see protocol10

  • Preculture

No.21,22:bcsA No.10:bcsB No.54,64:bcsC No.75:bcsD

procedure

refer to protocol10

Experiment:Sequence

material

  • PCR
    • DNA(No.21,22,10,54,64,75:following before "Miniprep")
    • Big Dye
    • primer
    • DW
  • Ethanol precipitation
    • ethanol
    • EDTA

procedure

  1. mix materials(DNA<50ng)
  2. PCR
  3. Add EDTA and Ethanol and put at room temperature　(15min)
  4. centrifuge (8000rpm,30min)
  5. throw away supernatant
  6. add ethanol again
  7. put at refrigerator
  8. throw away supernatant
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