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Team:Groningen/Notebook/4 September 2009
From 2009.igem.org
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| - | '''Purification''' | + | [[Image:Zymoclean Gel DNA Recovery Kit (D4001) 2.jpg|thumb|300px| www.sigmaaldrich.com]] |
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| + | '''Gel Purification''' | ||
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| + | * In step 7 an amount of 10μL MQ was added to elute the DNA fragments. | ||
===Transporters=== | ===Transporters=== | ||
Revision as of 08:27, 4 September 2009
[http://2009.igem.org/Team:Groningen http://2009.igem.org/wiki/images/f/f1/Igemhomelogo.png]
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Wet
GVP Cluster
[http://partsregistry.org/Part:BBa_F2620 BBa_F2620]
O.n. precultures
- → All four o.n. precultures showed bacterial growth, and could be used to isolate plasmid. The four are registry vector pSB1A2 with pLacI/pBad-araC and GVP. The isolated plasmid is used for transformation into pSB2K3 and sent for sequencing.
Plasmid Purification
Plasmid isolation was performed on the cultures of E.coli TOP10 containing the above mentioned plasmids 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 30μL MQ and stored in the fridge
Concentrations
| Plasmid | Conc. ng/μL | 260/280 | 260/230 | -20 box (michael | Restriction Control |
| pSB1A2 pLacI-GVP no.1 | 405.3 | 1.85 | 2.36 | D-6 | Yes (EcoRI/PstI) |
| pSB1A2 pLacI-GVP no.2 | 34.0 | 2.01 | 2.20 | D-7 | Yes (EcoRI/PstI) |
| pSB1A2 pBad/araC-GVP no.1 | 27.5 | 2.09 | 2.16 | D-8 | Yes (EcoRI/PstI) |
| pSB1A2 pBad/araC-GVP no.2 | 33.3 | 1.96 | 2.06 | D-9 | Yes (EcoRI/PstI) |
Restriction for Assembly
The vector [http://partsregistry.org/wiki/index.php?title=Part:pSB1A2 pSB1A2] containing the pLacI and pBad-araC with [http://partsregistry.org/Part:BBa_I750016 GVP] composite parts were cut with PstI and EcoRI to create correct ends for insert into [http://partsregistry.org/Part:pSB2K3 pSB2K3], which was also cut with EcoRI and PstI (4x) on 3-9.
| Plasmid | Amount μL | MQ μL | Fast digest buffer | EcoRI fast digest enzyme | XbaI fast digest enzyme | SpeI fast digest enzyme | PstI fast digest enzyme |
| pSB1A2 pLacI-GVP no.1 | 3.0 | 13.0 | 3.0 | 1.0 | x | x | 1.0 |
| pSB1A2 pLacI-GVP no.2 | 16.0 | x | 3.0 | 1.0 | x | x | 1.0 |
| pSB1A2 pBad/araC-GVP no.1 | 16.0 | x | 3.0 | 1.0 | x | x | 1.0 |
| pSB1A2 pBad/araC-GVP no.2 | 16.0 | x | 3.0 | 1.0 | x | x | 1.0 |
Restriction was kept at 37C for 40 min. and put on ice until used for gel purification.
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Gel Purification
- In step 7 an amount of 10μL MQ was added to elute the DNA fragments.
Transporters
Metal Accumulation
Vectors
Dry
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