Team:Tokyo Metropolitan/Project/Fiber/Protocol
From 2010.igem.org
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*Binding buffer | *Binding buffer | ||
*silica gel | *silica gel | ||
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*centrifuge | *centrifuge | ||
*vortex | *vortex | ||
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#Add 3 times Binding buffer than digestion production | #Add 3 times Binding buffer than digestion production | ||
#Add 10µl of silica gel and mix with Vortex | #Add 10µl of silica gel and mix with Vortex |
Revision as of 05:06, 26 October 2010
![](https://static.igem.org/mediawiki/2010/9/9f/Re-main.png)
E.coli Fiber Project Protocol
Protocol1:Grow up a culture of A.xylinum
Material
Equipment
Procedure
Note
|
Protocol2:Grow up a culture of E.coli
Material
Equipment
Procedure
|
Protocol3:PCR
Material
Equipment
Procedure
|
Protocol4:Agarose gel electrophoresis
Material
Equipment
Procedure
|
Protocol5:DNA purification from agarose gel
Material
Equipment
Procedure
|
Protocol6:Restriction enzyme digestion
Material
Equipment
Procedure
|
Protocol7:Purification
Material
- Binding buffer
- silica gel
- wash buffer
- TE buffer
- centrifuge
- vortex
- aspirator
- pipette
- pipette tip
- Add 3 times Binding buffer than digestion production
- Add 10µl of silica gel and mix with Vortex
- Centrifuge 1min
- Remove supernatant with aspirator
- Add Wash buffer and mix with vortex
- Centrifuge 30sec
- Remove supernatant with aspirator
- Remove ethanol by drying
- Add TE buffer and mix with Vortex
- Centrifuge 30sec
- DNA is solved in Supernatant
- 2×ligation Mix(Nippon gene)
- plasmid DNA
- insert DNA
- incubater
- PCR tube
- pipette
- freezer
- freezer box
- Incubate at room temperature for 5minutes
- E.coli Competent cell (JM109/NovaBlue)
- ligation production(refer to Ligation)
- autoclave
- incubator
- heater
- bunsen burner
- flask(500ml)
- plate
- tube
- pipette
- pipette tip
- ice
- add 50µl of E.coli Competent cells to tubes which was used ligation
- incubate the cells on ice for 30minutes
- heat shock the cells by heater at42°C for 45sec
- incubate the cells on ice for 2 min
- streak the cells on LB medium plate added chloramphenicol
- incubate cells at 37°C during for 14hours
- E.coli (plasmid)
- SolutionⅠ
- SolutionⅡ
- SolutionⅢ
- matrix
- TE buffer
- centrifuge
- microwave
- vortex mixer
- tube
- filter
- pick E.coli cells up from a colony and add to 2ml tube
- centrifuge 15000rpm/30sec and throw supernatant fluid away
- add 120µl of Solution I and vortex
- add 250µl of Solution II and shake with hand
- add 250µl of Solution III
- centrifuge 15000rpm/5min
- set spin filter in 2ml tube
- add supernatant to spin filter, then add 200µl of matrix and suspend with pipet
- centrifuge 15000rpm/30sec and throw supernatant fluid away
- add wash buffer 500µl and then centrifuge 15000rpm/30sec and throw supernatant fluid away (twice)
- centrifuge 15000rpm/2min and throw supernatant fluid away
- set spin filter in 1.5ml tube
- add TE buffer to 15ml falcon tube and heat 15min with microwave
- add 100µl of TE buffer
- centrifuge 15000rpm/1min
Protocol8:Ligation
Material
Equipment Procedure |
Protocol9:Transformation
Material
Equipment Procedure |
Protocol10:Miniprep (extraction of plasmid kit)
Material
Equipment Procedure |