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

E.coli Fiber Project Notebook

2010/10/24 Sunday (Naoto)

member

naoto and watachin

Experiment:Colony PCR

material

• colony of E.coli
  • bcsB:1~32
  • bcsC:33~64
  • bcsD:65~96
• other materials were same as protocol3

result

From the length of bands bcsC→No.54 and 64 bcsD→No.75 seem to be correct inserts

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

• Competent cell
  • Ecos JM109/Nova Blue

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