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Team:Groningen/Notebook/5 August 2009
From 2009.igem.org
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| - | ::* add 4 uL of the | + | ::* add 4 uL of the - ligation mixture to 50uL competent e.coli top10 cells. |
Incubate: | Incubate: | ||
:::*30 min @ ice | :::*30 min @ ice | ||
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::*add 800uL LB | ::*add 800uL LB | ||
:::* incubate for 1 h at 37° | :::* incubate for 1 h at 37° | ||
| - | ::* plate on LB-AMP plates | + | ::* plate on LB-AMP plates (one glpF 04-08 on chlorophenacol |
==Dry== | ==Dry== | ||
{{Team:Groningen/Notebook/Day/Footer}} | {{Team:Groningen/Notebook/Day/Footer}} | ||
Revision as of 13:14, 5 August 2009
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It's my birthday!!! (Michael)
So cake it is....
Wet
GVP Cluster
- → DONE 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
Transporters
GlpF Restriction, ligation and transformation as noted below.
Metal Accumulation
Restriction
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- 37 30min
- Inactivation on Gel
- Extraction from gel
Ligation
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- Tranformation
- add 4 uL of the - ligation mixture to 50uL competent e.coli top10 cells.
Incubate:
- 30 min @ ice
- 5 min 37°
- 5min @ ice
- add 800uL LB
- incubate for 1 h at 37°
- plate on LB-AMP plates (one glpF 04-08 on chlorophenacol
Dry
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