Team:Groningen/Notebook/5 August 2009
From 2009.igem.org
(Difference between revisions)
m (→GVP Cluster) |
(→GVP Cluster) |
||
Line 12: | Line 12: | ||
:→ {{todo}} isolate plasmids from overnight precultures | :→ {{todo}} isolate plasmids from overnight precultures | ||
:→ {{done}} perform a ligation between cut vector and GVP fragment | :→ {{done}} perform a ligation between cut vector and GVP fragment | ||
- | :→ {{ | + | :→ {{done}} transform E.coli TOP10 competent cells with ligation product |
'''Ligation''' | '''Ligation''' |
Revision as of 11:33, 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
- → DONE 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
|
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
|
|
|
|