From 2010.igem.org
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
- 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
- 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
- procedure
- mix materials(DNA<50ng)
- PCR
- Add EDTA and Ethanol and put at room temperature (15min)
- centrifuge (8000rpm,30min)
- throw away supernatant
- add ethanol again
- centrifuge (8000rpm,15min)
- put at refrigerator
- throw away supernatant
- add Hi-Di solution
- transfer these sample to plate for sequence
- read sequence