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Team:Groningen/Notebook/31 July 2009
From 2009.igem.org
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*Restiction/ligase and transformation of GlpF in psB1AC3 | *Restiction/ligase and transformation of GlpF in psB1AC3 | ||
| - | : | + | : '''Restiction''' |
{| | {| | ||
|10ul | |10ul | ||
| Line 33: | Line 33: | ||
| - | : | + | :'''Ligation''' |
{| | {| | ||
|2 uL | |2 uL | ||
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| - | + | :'''Tranformation''' | |
| - | :* add 4 uL of the GlpFpsB1AC3 ligation mixture to 50uL competent e.coli top10 cells. | + | ::* add 4 uL of the GlpFpsB1AC3 ligation mixture to 50uL competent e.coli top10 cells. |
Incubate: | Incubate: | ||
| - | ::*30 min @ ice | + | :::*30 min @ ice |
| - | ::*5 min 37° | + | :::*5 min 37° |
| - | ::*5min @ ice | + | :::*5min @ ice |
| - | :*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 |
===Metal Accumulation=== | ===Metal Accumulation=== | ||
Revision as of 12:38, 31 July 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
Transporters
GlpF
- Restiction/ligase and transformation of GlpF in psB1AC3
- Restiction
| 10ul | GlpF PCR |
| 2 ul | Buffer |
| 05.ul | EcoRI |
| 0.5 ul | SpeI |
| 7 uL | MQ |
- 37 30min
- Inactivation on Gel
- Extraction from gel
- Ligation
| 2 uL | Ligase buffer | 16° 1h → 65° 10 min | |
| 1 ul | T4 Ligase | ||
| 11 uL | GlpF digested with EcoRI and SpeI | 42° 2 min → | |
| 11 uL | psB1AC3 digested with EcoRi and SpeI |
- Tranformation
- add 4 uL of the GlpFpsB1AC3 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
Metal Accumulation
ArsR-MBP fusion
- colony PCR MBP:
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- Overnight culture MBP
Vectors
Dry
We had a look at ways to measure things concerning gas vesicles (buoyant density, volume, etc.), buoyant density was [http://dx.doi.org/10.1016/0167-7012(92)90048-9 previously measured] using a specific apparatus which we're not likely to obtain. We also talked with some biologists about ways to detect protein concentrations (or rather, amounts) using antibodies, apparently this might be possible if we attach something for which there is a kind of "standard" antibody available (and the maltose binding protein we're attaching to ArsR might qualify as such).
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