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Team:Groningen/Notebook/16 July 2009
From 2009.igem.org
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===GVP Cluster=== | ===GVP Cluster=== | ||
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| + | '''Colony PCR on cells containing the GVP and J23100 ligation product''' | ||
| + | |||
| + | * Colonies are picked from the plate with a sterile toothpick and resuspended in 20 μL MQ | ||
| + | * Mix (1): | ||
| + | - 2.5 μL Taq buffer (NH4) | ||
| + | - 0.2 μL dNTP | ||
| + | - 2 μL MgCl | ||
| + | - 16.3 μL MQ | ||
| + | - 1 μL VF2 primer | ||
| + | - 1 μL VR primer | ||
| + | - 1 μL colony solution | ||
| + | - 1 μL Taq | ||
| + | * Mix (2): | ||
| + | - 2.5 μL Taq buffer (KCl) | ||
| + | - 0.2 μL dNTP | ||
| + | - 1.5 μL MgCl | ||
| + | - 16.8 μL MQ | ||
| + | - 1 μL VF2 primer | ||
| + | - 1 μL VR primer | ||
| + | - 1 μL colony solution | ||
| + | - 1 μL Taq | ||
| + | |||
| + | {| | ||
| + | ! PCR program | ||
| + | ! Temperature | ||
| + | ! Time | ||
| + | |- | ||
| + | |Denaturing | ||
| + | |95° | ||
| + | |5.00 min | ||
| + | |- | ||
| + | | | ||
| + | |Start Cycles 20X | ||
| + | |- | ||
| + | |Denaturing | ||
| + | |95° | ||
| + | |30 sec | ||
| + | |- | ||
| + | |Annealing | ||
| + | |55° | ||
| + | |30 sec | ||
| + | |- | ||
| + | |Elongation | ||
| + | |72° | ||
| + | |6.10 min | ||
| + | |- | ||
| + | | | ||
| + | |End cycles | ||
| + | |- | ||
| + | |Hold | ||
| + | |4° | ||
| + | |Forever | ||
| + | |} | ||
| + | |||
| + | * PCR products were analysed on gel (1% agarose, TBE, EtBr, 25 min, 100V) | ||
| + | |||
| + | Results: No bands | ||
| + | |||
| + | '''Inoculation of Colonies for restriction analysis''' | ||
| + | |||
| + | * 3 ml Ty (100 μg/ml Ampicillin) inoculated with colonies from plate (with sterile toothpick) | ||
| + | * Incubation o/n 37°C in shaker | ||
| + | |||
===Transporters=== | ===Transporters=== | ||
Revision as of 10:37, 17 July 2009
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Wet
GVP Cluster
Colony PCR on cells containing the GVP and J23100 ligation product
- Colonies are picked from the plate with a sterile toothpick and resuspended in 20 μL MQ
- Mix (1):
- 2.5 μL Taq buffer (NH4) - 0.2 μL dNTP - 2 μL MgCl - 16.3 μL MQ - 1 μL VF2 primer - 1 μL VR primer - 1 μL colony solution - 1 μL Taq
- Mix (2):
- 2.5 μL Taq buffer (KCl) - 0.2 μL dNTP - 1.5 μL MgCl - 16.8 μL MQ - 1 μL VF2 primer - 1 μL VR primer - 1 μL colony solution - 1 μL Taq
| PCR program | Temperature | Time |
|---|---|---|
| Denaturing | 95° | 5.00 min |
| Start Cycles 20X | ||
| Denaturing | 95° | 30 sec |
| Annealing | 55° | 30 sec |
| Elongation | 72° | 6.10 min |
| End cycles | ||
| Hold | 4° | Forever |
- PCR products were analysed on gel (1% agarose, TBE, EtBr, 25 min, 100V)
Results: No bands
Inoculation of Colonies for restriction analysis
- 3 ml Ty (100 μg/ml Ampicillin) inoculated with colonies from plate (with sterile toothpick)
- Incubation o/n 37°C in shaker
Transporters
We decided to make MasterMixes of our own. All but 1uL template, 2uL primers and 1uL taq for 25uL reactions.
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Metal Accumulation
Vectors
Dry
KB extended the Simbiology model to support OpG and OpH in addition to OpN. Jasper in the mean time added some more explanation to the modelling section on the metal accumulation page.
In the end of the afternoon we tried to find more modelling software. Likely candidates are SEMPPR (Japer) and cytoscape (KB) Furthermore KB is trying to get up to date with the paper knowledge.
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