"
Team:Tokyo Metropolitan/Notebook/Fiber/2010/10/24
From 2010.igem.org
(Difference between revisions)
| Line 79: | Line 79: | ||
#throw away supernatant | #throw away supernatant | ||
#add ethanol again | #add ethanol again | ||
| + | #centrifuge (8000rpm,15min) | ||
#put at refrigerator | #put at refrigerator | ||
#throw away supernatant | #throw away supernatant | ||
| + | #add Hi-Di solution | ||
| + | #transfer these sample to plate for sequence | ||
| + | #read sequence | ||
<br/> | <br/> | ||
Revision as of 16:15, 25 October 2010
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
- 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
