From 2009.igem.org
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| + | Plasmid Isolation of luxOD47A in pCR2.1 TOPO and psB1AC3 vectors |
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| - | WIKI CODING HERE
| + | The purpose was to isolate plasmid containing luxOD47A, as a means of starting the construction of the mutant circuits. TOPO T/A and psB1AC3 vectors containing luxOD47A were isolated using the Sigma GenElute Plasmid Mini-prep kit. 100μL of ddH2O were used to elute, and the purity and concentration of each plasmid were measured using the NanoDrop Spectrophotometer. |
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Revision as of 23:53, 3 August 2009
University of Calgary
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CAROL
Plasmid Isolation of ''luxCDABE'' in TOPO vector
The purpose of plasmid isolation is to extract plasmids from E. coli that has topo vector with luxCDABE. Overnight cultures were prepared and Sigma Mini Prep kit was used to to isolate plasmids (see protocols for detailed procedures). The plasmids was finally eluted in the elution buffer provided by the kitto a total of 50uL.
The concentration of plasmids was determined using the nano-drop apparatus at 260nm wavelength (see protocols for detailed procedures) and the following table summarizes the concentrations of plasmids.
| Plasmid | 260/280 | 260/230 | Concentration [ng/μL]
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| luxCDABE TOPO C1 | 2.21 | 4.58 | 47.6
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| luxCDABE TOPO C2 | 2.09 | 3.39 | 56.0
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CHINMOYEE
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EMILY
Plasmid Isolation of LuxOD47E in pCR2.1 TOPO and psB1AC3 vectors
- Objective: To extract plasmids of LuxOD47E in pCR2.1-TOPO vector from overnight cultures.
- Protocol: Used Sigma GenElute Plasmid Miniprep Kit (Sigma), lot # 048k6062, used as according to the manufacturers instructions, eluting in 50 μL ddH20.
- Results: Used Nanodrop 1000 Spectrophotometer read at 260 wavelength to determine DNA concentrations
| Plasmid | 260/280 | 260/230 | Concentration [ng/μL]
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| LuxOD47E TOPO C1 | 1.88 | 2.21 | 409.5
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| LuxOD47E TOPO C2 | 1.86 | 2.18 | 551.4
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| LuxOD47E BBK C1 | 2.04 | 5.01 | 551.4
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| LuxOD47E BBK C2 | 1.77 | 1.39 | 92.9
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FAHD
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IMAN
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JAMIE
Plasmid Isolation of luxOU in pCR-Blunt II-TOPO and psB1AC3
Sigma Genelute Plasmid Mini-prep kit (lot # 048k6062)as used. Standard manufacturer protocol used; elution in 50µL of ddH 2O.
| Plasmid sample |
260/280 |
260/230 |
Concentration(ng/µL) |
| TOP10 luxOU C1 |
1.91 |
2.58 |
365.6 |
| TOP10 luxOU C2 |
1.90 |
2.52 |
287.2 |
| BBK luxOU C1 |
1.87 |
2.22 |
534.2 |
| BBK luxOU C2 |
1.92 |
2.24 |
715.2 |
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JEREMY
Plasmid Isolation of luxOD47A in pCR2.1 TOPO and psB1AC3 vectors
The purpose was to isolate plasmid containing luxOD47A, as a means of starting the construction of the mutant circuits. TOPO T/A and psB1AC3 vectors containing luxOD47A were isolated using the Sigma GenElute Plasmid Mini-prep kit. 100μL of ddH2O were used to elute, and the purity and concentration of each plasmid were measured using the NanoDrop Spectrophotometer.
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KATIE
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KEVIN
Plasmid Isolation of Pqrr4 for Reporter circuit
The Pqrr4 was isolated from TOPO T/A and biobrick AC (pSB1AC3) using Sigma’s Genelute Plasmid Mini-prep kit (sigma) according to the specifications of the manufacturer. Nanodrop utility along with the Spectrophotometer was used to measure the concentration and purity of the isolated plasmid. The wavelength was set at 260nm.
HTML Code, just as an example for now
| Plasmid | 260/280 | 260/230 | Concentration [ng/μL]
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| Pqrr4 TOPO Blue tube | 1.81 | 2.24 | 181
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| Pqrr4 TOPO Pink tube | 1.81 | 2.02 | 231.3
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| Pqrr4 BBK Green tube | 1.76 | 1.66 | 51.2
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| Pqrr4 BBK Pink tube | 1.85 | 1.45 | 60.6
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*The two TOPO vectors were from the same colony, thus labelled "Green tube" instead of C1. The two BBK plasmids were also from same colony, so same label applies.
Wiki Code
Table 1. Purity and concentration of Pqrr4 measured with Nanodrop utility and Spectrophotometer
| Plasmid | 260/280 | 260/230 | Concentration [ng/μL]
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| Pqrr4 TOPO Blue Tube | 1.81 | 2.24 | 181
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| Pqrr4 TOPO Pink Tube | 1.81 | 2.02 | 231.3
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| Pqrr4 BBK Green Tube | 1.76 | 1.66 | 51.2
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| Pqrr4 BBK Pink Tube | 1.85 | 1.45 | 60.6
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MANDY
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PATRICK
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PRIMA
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STEFAN
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VICKI
Plasmid isolation with mini-prep
Purpose:
To isolate the plasmid DNA from the rest of the bacteria for colonies containing LuxPQ on TOPO II Blunt and LuxPQ BBk on psB1AC3.
Reference: The user manual and the Sigma Aldrich site
Kit: MP70 GeneElute Plasmid mini-prep kit. Note that it had been previously used, which may be a problem for contamination.
Materials and methods:
Procedure was carried out in accordance with our Plasmid Isolation protocol. When complete, we stored our isolated plasmids in the freezer for concentration measurement and content analysis over the next 2 days.
Results:
We were left with tubes containing the described plasmids. Quantifiable details on their concentrations and actual contents are outlined on the May 27 and May 28 entries.
DNA concentration measurements with the Nanodrop spectrophotometer
Purpose:
To measure the DNA concentration of LuxPQ plasmids that were isolated in the May 26 miniprep
Materials and Methods:
The DNA measurement area was cleaned with isopropanol and KimWipes to clear off any trace residue from previous uses. The sample wavelength was set to 260 nm. A blank measurement was made with 1 uL of ddHOH. 1 uL of the sample in question was added to the Nanodrop measurement area. The measurement was made; the sample area was wiped down; and a new sample was measured.
Results:
| Plasmid | 260/280 | 260/230 | Concentration [ng/μL]
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| LuxPQ TOPO C1 | 1.83 | 1.86 | 338.2
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| LuxPQ TOPO C2 | 1.81 | 1.87 | 284.2
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| LuxPQ BBK C1 | 1.90 | 2.34 | 295.5
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| LuxPQ BBK C2 | 1.90 | 2.29 | 313.2
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