Team:Groningen/Notebook/17 August 2009
From 2009.igem.org
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m (→GVP Cluster) |
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===GVP Cluster=== | ===GVP Cluster=== | ||
- | :→ {{ | + | :→ {{done}} Restriction for Assembly |
:→ {{todo}} Gel purification of plasmid | :→ {{todo}} Gel purification of plasmid | ||
:→ {{todo}} Ligation of metal promoter oligo's into vector BBa_J61002 | :→ {{todo}} Ligation of metal promoter oligo's into vector BBa_J61002 | ||
:→ {{todo}} Transformation of E.coli TOP10 cells | :→ {{todo}} Transformation of E.coli TOP10 cells | ||
:→ {{todo}} Test promoter/GVP constructs (grow precultures) | :→ {{todo}} Test promoter/GVP constructs (grow precultures) | ||
- | :→ {{todo}} Grow o.n. cultures to check ligation of pSB3K3-H/L-GVP constructs of last week | + | :→ {{todo}} Grow o.n. cultures to check ligation of pSB3K3-H/L-GVP constructs of last week (3 each) |
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The vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] containing the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 high] constitutive promoter was cut with EcoRI and SpeI to create correct ends for insert of metal promoter oligo's. | The vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] containing the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 high] constitutive promoter was cut with EcoRI and SpeI to create correct ends for insert of metal promoter oligo's. | ||
+ | |||
+ | :→ Restriction in this way should cut out the original promoter, but the fragment is small and hard to detect on gel. The different size between double cut and single cut vector is to small to separate, and a bit of luck is needed to avoid self ligation of original promoter into vector. | ||
* 8 μL plasmid in MQ (1.0μg) | * 8 μL plasmid in MQ (1.0μg) |
Revision as of 10:20, 17 August 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
- → DONE Restriction for Assembly
- → TODO Gel purification of plasmid
- → TODO Ligation of metal promoter oligo's into vector BBa_J61002
- → TODO Transformation of E.coli TOP10 cells
- → TODO Test promoter/GVP constructs (grow precultures)
- → TODO Grow o.n. cultures to check ligation of pSB3K3-H/L-GVP constructs of last week (3 each)
Restriction for Assembly
The vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] containing the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 high] constitutive promoter was cut with EcoRI and SpeI to create correct ends for insert of metal promoter oligo's.
- → Restriction in this way should cut out the original promoter, but the fragment is small and hard to detect on gel. The different size between double cut and single cut vector is to small to separate, and a bit of luck is needed to avoid self ligation of original promoter into vector.
- 8 μL plasmid in MQ (1.0μg)
- 8 μL MQ (end volume of 20μL)
- 2μL Fast digest buffer
- 1μL PstI fast digest enzyme
- 1μL SpeI fast digest enzyme
Restriction was kept at 37C for 30 min. and put on ice until used for gel purification.
Purification
Concentrations
Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
BBa_J61002-H SpeI/EcoRI | ? | ? | ? | ? | ? |
Ligation
Tranformation
Transporters
Metal Accumulation
Vectors
Dry
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