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Team:Groningen/Notebook/9 July 2009
From 2009.igem.org
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(New page: {{Team:Groningen/Notebook/Day/Header}} ==Wet== '''Purification of HmtA (PA Q9I147 6xHis Tag)''' * SDS-gel electrophosis * Make a 12% SDS-gel * Fill gel with samples Wash 1-2 20mM imidazo...) |
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==Wet== | ==Wet== | ||
| + | ===GVP Cluster=== | ||
| + | '''Inoculation of TY-medium for Glycerol Stocks and Plasmid isolation''' | ||
| + | |||
| + | * [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109] (labelled as no.1) vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] in ''E.coli TOP10'' | ||
| + | * [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] (labelled as no.2) vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] in ''E.coli TOP10'' (red colony) | ||
| + | * [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106] (labelled as no.3) vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] in ''E.coli TOP10'' (red colony) | ||
| + | * The three plates showing new colonies were used to inoculate 5mL TY-medium with amp. (final conc. 100mg/ul). | ||
| + | * The tubes were put in a waterbath at 37C (200 rpm) and grown o.n. | ||
| + | |||
| + | ===Transporters=== | ||
| + | [[Image:HmtA_SDS_gel.jpg|200px|thumb|right|[Team:Groningen/Team|First results!]] | ||
'''Purification of HmtA (PA Q9I147 6xHis Tag)''' | '''Purification of HmtA (PA Q9I147 6xHis Tag)''' | ||
* SDS-gel electrophosis | * SDS-gel electrophosis | ||
* Make a 12% SDS-gel | * Make a 12% SDS-gel | ||
* Fill gel with samples Wash 1-2 20mM imidazole, Wash1-2 40mM imidazole, Elute1-3 and ladders | * Fill gel with samples Wash 1-2 20mM imidazole, Wash1-2 40mM imidazole, Elute1-3 and ladders | ||
| - | * Run | + | * Run 2h at 100V |
| + | * Stain the gel in Coomassie blue (2h) | ||
| + | * Destain with demiwater (2x, 10 min microwave) | ||
| + | |||
| + | |||
| + | |||
| + | |||
| + | ==Vectors== | ||
| + | Measure concentration with Nanodrop | ||
| + | |||
| + | {| | ||
| + | | | ||
| + | <!--Tabel 1 hier--> | ||
| + | |||
| + | {| border="1" | ||
| + | |- | ||
| + | ! Component | ||
| + | ! concentrations ng/ul | ||
| + | ! 260/280 | ||
| + | |- | ||
| + | ! pSB1AC3 | ||
| + | | 205,7 | ||
| + | |1,91 | ||
| + | |- | ||
| + | ! pSBK3 | ||
| + | | 24,4 | ||
| + | |2,05 | ||
| + | |} | ||
| + | ==Dry== | ||
| + | A lot of time was spent on finding dissociation constants and figuring out how ArsR/D work, and thinking of how to model their interaction. An input function was derived for the ArsR/D operator, but it is not yet completely clear how this should be used in a model (it is a function of the ArsR/D concentration, but at the same time produces ArsR/ArsD). It would probably be better to start with As(III) and derive an input function from that, if at all possible. | ||
{{Team:Groningen/Notebook/Day/Footer}} | {{Team:Groningen/Notebook/Day/Footer}} | ||
Latest revision as of 12:12, 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
Inoculation of TY-medium for Glycerol Stocks and Plasmid isolation
- [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23109 BBa_J23109] (labelled as no.1) vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] in E.coli TOP10
- [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23100 BBa_J23100] (labelled as no.2) vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] in E.coli TOP10 (red colony)
- [http://partsregistry.org/wiki/index.php?title=Part:BBa_J23106 BBa_J23106] (labelled as no.3) vector [http://partsregistry.org/wiki/index.php?title=Part:BBa_J61002 BBa_J61002] in E.coli TOP10 (red colony)
- The three plates showing new colonies were used to inoculate 5mL TY-medium with amp. (final conc. 100mg/ul).
- The tubes were put in a waterbath at 37C (200 rpm) and grown o.n.
Transporters
Purification of HmtA (PA Q9I147 6xHis Tag)
- SDS-gel electrophosis
- Make a 12% SDS-gel
- Fill gel with samples Wash 1-2 20mM imidazole, Wash1-2 40mM imidazole, Elute1-3 and ladders
- Run 2h at 100V
- Stain the gel in Coomassie blue (2h)
- Destain with demiwater (2x, 10 min microwave)
Vectors
Measure concentration with Nanodrop
DryA lot of time was spent on finding dissociation constants and figuring out how ArsR/D work, and thinking of how to model their interaction. An input function was derived for the ArsR/D operator, but it is not yet completely clear how this should be used in a model (it is a function of the ArsR/D concentration, but at the same time produces ArsR/ArsD). It would probably be better to start with As(III) and derive an input function from that, if at all possible.
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