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

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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
see 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. centrifuge (8000rpm,15min)
  8. put at refrigerator
  9. throw away supernatant
  10. add Hi-Di solution
  11. transfer these sample to plate for sequence
  12. read sequence