Team:Groningen/Notebook/19 August 2009
From 2009.igem.org
(Difference between revisions)
(New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== ===GVP Cluster=== :→ {{todo}} Restriction for Assembly :→ {{todo}} Gel purification of plasmid/insert :→ {{todo}} Ligation of GVP in...) |
(→GVP Cluster) |
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Line 10: | Line 10: | ||
:→ {{todo}} Transformation of E.coli TOP10 cells (kan.) | :→ {{todo}} Transformation of E.coli TOP10 cells (kan.) | ||
:→ {{todo}} Test promoter/GVP constructs (grow precultures) | :→ {{todo}} Test promoter/GVP constructs (grow precultures) | ||
+ | |||
+ | '''Restriction for Assembly''' | ||
+ | |||
+ | The vector [http://partsregistry.org/wiki/index.php?title=Part:pSB3K3 pSB3K3] containing the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 low] constitutive promoter was cut with PstI and SpeI to create correct ends for insert of GVP biobrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 BBa_I750016], which was cut with XbaI and PstI. | ||
+ | |||
+ | * 16μL pSB3K3-L in MQ (0.8μg) | ||
+ | * 2μL Fast digest buffer | ||
+ | * 1μL PstI fast digest enzyme | ||
+ | * 1μL SpeI fast digest enzyme | ||
+ | |||
+ | * 10μL plasmid-GVP in MQ (?μg) | ||
+ | * 6μL MQ (end volume of 20μL) | ||
+ | * 2μL Fast digest buffer | ||
+ | * 1μL PstI fast digest enzyme | ||
+ | * 1μL SpeI/XbaI fast digest enzyme | ||
+ | |||
+ | Restriction was kept at 37C for 30 min. and put on ice until used for gel purification. | ||
+ | |||
+ | '''Purification''' | ||
+ | |||
+ | '''Concentrations''' | ||
+ | |||
+ | {|cellpadding="2" cellspacing="1" border="4" | ||
+ | |'''Plasmid''' | ||
+ | |'''Conc. ng/μL''' | ||
+ | |'''260/280 | ||
+ | |'''260/230 ''' | ||
+ | |'''-20 box (michael | ||
+ | |'''Restriction Control''' | ||
+ | |- | ||
+ | |pSB3K3-L (SpeI/PstI) | ||
+ | |? | ||
+ | |? | ||
+ | |? | ||
+ | |? | ||
+ | |? | ||
+ | |- | ||
+ | |GVP (XbaI/PstI) | ||
+ | |? | ||
+ | |? | ||
+ | |? | ||
+ | |? | ||
+ | |? | ||
+ | |} | ||
+ | |||
+ | '''Restriction Control''' | ||
===Transporters=== | ===Transporters=== |
Revision as of 10:25, 19 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
- → TODO Restriction for Assembly
- → TODO Gel purification of plasmid/insert
- → TODO Ligation of GVP into vector pSB3K3
- → TODO Transformation of E.coli TOP10 cells (kan.)
- → TODO Test promoter/GVP constructs (grow precultures)
Restriction for Assembly
The vector [http://partsregistry.org/wiki/index.php?title=Part:pSB3K3 pSB3K3] containing the [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 low] constitutive promoter was cut with PstI and SpeI to create correct ends for insert of GVP biobrick [http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 BBa_I750016], which was cut with XbaI and PstI.
- 16μL pSB3K3-L in MQ (0.8μg)
- 2μL Fast digest buffer
- 1μL PstI fast digest enzyme
- 1μL SpeI fast digest enzyme
- 10μL plasmid-GVP in MQ (?μg)
- 6μL MQ (end volume of 20μL)
- 2μL Fast digest buffer
- 1μL PstI fast digest enzyme
- 1μL SpeI/XbaI 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 |
pSB3K3-L (SpeI/PstI) | ? | ? | ? | ? | ? |
GVP (XbaI/PstI) | ? | ? | ? | ? | ? |
Restriction Control
Transporters
Metal Accumulation
Vectors
Dry
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