Team:Groningen/Notebook/5 August 2009
From 2009.igem.org
(Difference between revisions)
(→GVP Cluster) |
m (→GVP Cluster) |
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Line 11: | Line 11: | ||
:→ {{todo}} buy a nice cake and eat it ;) | :→ {{todo}} buy a nice cake and eat it ;) | ||
:→ {{todo}} isolate plasmids from overnight precultures | :→ {{todo}} isolate plasmids from overnight precultures | ||
- | :→ {{ | + | :→ {{done}} perform a ligation between cut vector and GVP fragment |
:→ {{todo}} transform E.coli TOP10 competent cells with ligation product | :→ {{todo}} transform E.coli TOP10 competent cells with ligation product | ||
'''Ligation''' | '''Ligation''' | ||
- | :*2 uL Ligase buffer | + | :* 2 uL Ligase buffer |
- | :*1 ul T4 Ligase | + | :* 1 ul T4 Ligase |
- | :* | + | :* 8 uL plasmid pSB1AC3 digested with PstI and SpeI |
- | :* | + | :* 4 uL insert GVP restricted with XbaI and PstI |
''Incubate:'' | ''Incubate:'' | ||
- | :* | + | :* 25°C 30min. |
- | :* | + | :* kept on ice for 10min. |
'''Tranformation''' | '''Tranformation''' | ||
- | :* add | + | :* add 10uL of the pSB1AC3-GVP ligation product to 50uL competent E.coli TOP10 cells. |
''Incubate:'' | ''Incubate:'' | ||
:* 30 min @ ice | :* 30 min @ ice | ||
- | :* | + | :* 50 sec 42°C |
- | :* | + | :* 2 min @ ice |
:* add 800uL LB-medium | :* add 800uL LB-medium | ||
- | :* incubate for 1 h at | + | :* incubate for 1 h at 37°C |
- | :* plate on LB- | + | :* plate on LB-amp<sub>50</sub> plates |
==Dry== | ==Dry== | ||
{{Team:Groningen/Notebook/Day/Footer}} | {{Team:Groningen/Notebook/Day/Footer}} |
Revision as of 10:01, 5 August 2009
It's my birthday!!! (Michael)
So cake it is....
Wet
GVP Cluster
- → TODO buy a nice cake and eat it ;)
- → TODO isolate plasmids from overnight precultures
- → DONE perform a ligation between cut vector and GVP fragment
- → TODO transform E.coli TOP10 competent cells with ligation product
Ligation
- 2 uL Ligase buffer
- 1 ul T4 Ligase
- 8 uL plasmid pSB1AC3 digested with PstI and SpeI
- 4 uL insert GVP restricted with XbaI and PstI
Incubate:
- 25°C 30min.
- kept on ice for 10min.
Tranformation
- add 10uL of the pSB1AC3-GVP ligation product to 50uL competent E.coli TOP10 cells.
Incubate:
- 30 min @ ice
- 50 sec 42°C
- 2 min @ ice
- add 800uL LB-medium
- incubate for 1 h at 37°C
- plate on LB-amp50 plates
Dry
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