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Team:Groningen/Notebook/7 August 2009
From 2009.igem.org
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===Metal Accumulation=== | ===Metal Accumulation=== | ||
| + | *'''2nd PCR to amplify SmtA and GST-SmtA''' | ||
| + | **Use pGEX-3X-GST-SmtA to amplify SmtA-GST and SmtA | ||
| + | **Use pET29a-SmtA to amplify SmtA | ||
| + | **Program: MT NIENKE (Left PCR machine) | ||
| + | ***Globally: 10cycles touchdown from 65-50°C, 25 cycles with Tm gradient from60-50°C (4steps), elongation time of 1:20. | ||
| + | **Mix | ||
| + | ::MM NH4 21uL | ||
| + | ::Primer fw [10mM] 1uL | ||
| + | ::Primer rev [10mM] 1uL | ||
| + | ::Vector 1uL | ||
| + | ::HomeTaq pol 1uL | ||
| + | **Run program o/n | ||
| + | **Check on gel | ||
===Vectors=== | ===Vectors=== | ||
Revision as of 10:12, 7 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 isolate plasmids from overnight precultures
- → TODO perform a restriction on isolated plasmid (should result in 6000 and 3000bp fragments)
- → TODO plate on cultures for pure colony over the weekend
- → TODO cut pSB3K3 plasmid and GVP containing plasmid for ligation
- → TODO purify wanted fragments
Plasmid Purification
Plasmid isolation was performed on the cultures of E.coli TOP10 containing plasmids [http://partsregistry.org/wiki/index.php?title=Part:pSB1AC3 pSB1AC3] with high, medium and low constitutive promoters and [http://partsregistry.org/wiki/index.php?title=Part:BBa_I750016 GVP] with the "Sygma-Aldrich™ [http://www.sigmaaldrich.com/life-science/molecular-biology/dna-and-rna-purification/plasmid-miniprep-kit.html GenElute™] Plasmid Miniprep Kit".
- From each tube 4mL of culture was collected in a 2.0mL cup, and the cells were pelleted by centrifugation for 1 min. at max. speed and the supernatant discarded.
- Plasmids were eluted with 20μL MQ and stored in the fridge
Concentration of Plasmids
<left>
| Component | Description | Part or Accession # | Base Pairs (bp) | Plasmid (backbone) |
| GVP | Gas Vesicle Proteins | [http://partsregistry.org/Part:BBa_I750016 BBa_I750016] | 6064 | [http://partsregistry.org/Part:BBa_J61035 BBa_J61035] |
Transporters
Metal Accumulation
- 2nd PCR to amplify SmtA and GST-SmtA
- Use pGEX-3X-GST-SmtA to amplify SmtA-GST and SmtA
- Use pET29a-SmtA to amplify SmtA
- Program: MT NIENKE (Left PCR machine)
- Globally: 10cycles touchdown from 65-50°C, 25 cycles with Tm gradient from60-50°C (4steps), elongation time of 1:20.
- Mix
- MM NH4 21uL
- Primer fw [10mM] 1uL
- Primer rev [10mM] 1uL
- Vector 1uL
- HomeTaq pol 1uL
- Run program o/n
- Check on gel
Vectors
Dry
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