Team:Groningen/Notebook/5 August 2009
From 2009.igem.org
(Difference between revisions)
m (→GVP Cluster) |
(→GVP Cluster) |
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:→ {{todo}} perform a ligation between cut vector and GVP fragment | :→ {{todo}} 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''' | ||
+ | |||
+ | :*2 uL Ligase buffer | ||
+ | :*1 ul T4 Ligase | ||
+ | :*20 uL plasmid pSB1AC3 digested with PstI and SpeI | ||
+ | :*6 uL insert GVP restricted with XbaI and PstI | ||
+ | |||
+ | ''Incubate:'' | ||
+ | :*16°C 60min. | ||
+ | :*65°C 10min. | ||
+ | |||
+ | '''Tranformation''' | ||
+ | :* add 15 uL of the pSB1AC3-GVP ligation product to 50uL competent E.coli TOP10 cells. | ||
+ | ''Incubate:'' | ||
+ | :* 30 min @ ice | ||
+ | :* 5 min 37°C | ||
+ | :* 5min @ ice | ||
+ | :* add 800uL LB-medium | ||
+ | :* incubate for 1 h at 37° | ||
+ | :* plate on LB-AMP plates | ||
==Dry== | ==Dry== | ||
{{Team:Groningen/Notebook/Day/Footer}} | {{Team:Groningen/Notebook/Day/Footer}} |
Revision as of 07:35, 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
- → TODO 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
- 20 uL plasmid pSB1AC3 digested with PstI and SpeI
- 6 uL insert GVP restricted with XbaI and PstI
Incubate:
- 16°C 60min.
- 65°C 10min.
Tranformation
- add 15 uL of the pSB1AC3-GVP ligation product to 50uL competent E.coli TOP10 cells.
Incubate:
- 30 min @ ice
- 5 min 37°C
- 5min @ ice
- add 800uL LB-medium
- incubate for 1 h at 37°
- plate on LB-AMP plates
Dry
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