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

E.coli Fiber Project Notebook

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