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Team:Groningen/Notebook/10 September 2009
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Wet
GVP Cluster
O.n. precultures
- → All tubes for plasmid isolation (pLacI (pSB1A2)(no.1 and no.2), pSB2K3 (12J)(2x) and pSB2K3 (12L)(2x) showed growth. The pSB2K3 had less growth (and smaller pellet) than the other tubes.
Plasmid isolation
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 (tubes from pSB2K3 12J and 12L were combined), 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 |
| pLacI no.1 (pSB1A2) | 66.3 | 1.86 | 1.82 | x | Yes (SpeI/PstI) |
| pLacI no.2 (pSB1A2) | 47.1 | 1.82 | 1.45 | x | Yes (SpeI/PstI) |
| pSB2K3 (12J) | 142.9 | 1.89 | 2.31 | x | Yes (EcoRI/PstI) |
| pSB2K3 (12L) | 337.1 | 1.87 | 2.35 | x | Yes (EcoRI/PstI) |
Restriction for Assembly
The plasmids from the o.n. precultures were cut with PstI and EcoRI to cut out the entire part between the pre- and suffix.
| Plasmid | Amount μL | MQ μL | Fast digest buffer | EcoRI fast digest enzyme | XbaI fast digest enzyme | SpeI fast digest enzyme | PstI fast digest enzyme |
| pLacI no.1 (pSB1A2) | 16.0 | x | 3.0 | x | x | 1.0 | 1.0 |
| pLacI no.1 (pSB1A2) | 16.0 | x | 3.0 | x | x | 1.0 | 1.0 |
| GVP | 4.0 | 11.0 | 3.0 | x | 1.0 | x | 1.0 |
| pSB2K3 (12J) | 8.0 | 6.0 | 3.0 | 1.0 | x | x | 1.0 |
| pSB2K3 (12L) | 4.0 | 11.0 | 3.0 | 1.0 | x | x | 1.0 |
| pZntR-GVP | 12.0 | 3.0 | 3.0 | 1.0 | x | x | 1.0 |
- → From left to right: 1kB ladder, pZntR-GVP (2x), Empty Slot, pSB2K3 12J (2x), Empty Slot, pSB2K3 12L (2x)
- → From left to right: 1kB ladder, pLacI no.1 (pSB1A2)(2x), pLacI no.2 (pSB1A2)(2x), GVP (2x), Empty Slot,
Purification
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- → In step 7 the fragments were eluted in 12μL MQ and was stored on ice until use.
Concentration
Ligation
A total amount of vector of 100ng was used in a 1:3 ratio with insert.
- 3 uL Ligase buffer
- 1 ul T4 Ligase
- uL plasmid pSB1A2 pLacI digested with SpeI and PstI
- uL GVP restricted with XbaI and PstI
- 3 uL Ligase buffer
- 1 ul T4 Ligase
- uL plasmid pSB2K3 digested with EcoRI and PstI
- uL pZntR-GVP and pCueO-GVP restricted with EcoRI and PstI
- uL MQ
Incubate:
- 25°C 50min.
- kept on ice for 10min.
Tranformation
- add 10uL of the ligation product to 50uL competent E.coli TOP10 cells.
Incubate:
- 30 min @ ice
- 90 sec 42°C
- 2 min @ ice
- add 800uL LB-medium
- incubate for 1 h at 37°C
- plate on LB-amp100 plates
- → Positive control was BBa_J61002-J23101 plasmid, and negative control was MQ.
Observations
Here are the allignments of the expect
Transporters
Metal Accumulation
Vectors
Dry
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