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- + Chloride Assay
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A method based on that of Bergmann and Sanik (Bergmann and Sanik, 1957) was used for quantitation of inorganic chloride, as follows. Resting cell supernatant (1 mL) was mixed with 200 µL Iron Reagent and 400 µL Mercury Reagent, incubated 10 min at room temperature, and the absorbance read at 460 nm. Chloride concentration was calculated via reference to a standard curve based on NaCl solutions in KP buffer (0, 0.1, 0.2, 0.5, 1.0 mM). Due to the time sensitivity of this assay, standards were prepared alongside every set of test samples, and processed at the same time. Test samples were diluted where necessary to bring them within the absorbance range of the standard curve (A460 approximately 0.045 – 1.080). The whole experiment was repeated three times, and the stoichiometry of chloride release calculated for each experiment as the number of moles Cl- produced per mole of chlorinated substrate degraded. The stoichiometry values from three independent experiments were averaged and statistical analysis were carried out using GraphPad Prism software using the one-way ANOVA (and nonparametric) test.
- • We have a stock solution of NaCl in KP buffer at our bench in the lab (but this could easily be made up), and Hugh has also made up some different solutions for the reference curve.
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• There are (up to) six vials that will be tested, these are in the cool room. Two are controls, which have E. Coli cells transformed with pBBR, two have E. Coli transformed with pBS-ToMO (toluene-o-xylene monooxygenase) and two have E. Coli transformed with pBS-TOM (toluene ortho-monooxygenase). There are two of each because one vial has DCA, the other doesn’t.
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• From our GC results last week, it appeared that only ToMO attacked DCA. [RESULTS FROM GC - pBBR(DCA) and TOM(DCA) had an area of ~500,000 at 2.8 minutes, whereas ToMO(DCA) had an area of ~280,000 at 2.8 minutes. ToMO(control) had something like 30,000 at 2.1 minutes. The results are in the lab-book and the vials are in the cool room. Every run is saved with a new file name on the GC computer except the ToMO(control), which I accidentally overrode.]
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A method based on that of Bergmann and Sanik (Bergmann and Sanik, 1957) was used for quantitation of inorganic chloride, as follows. Resting cell supernatant (1 mL) was mixed with 200 µL Iron Reagent and 400 µL Mercury Reagent, incubated 10 min at room temperature, and the absorbance read at 460 nm. Chloride concentration was calculated via reference to a standard curve based on NaCl solutions in KP buffer (0, 0.1, 0.2, 0.5, 1.0 mM). Due to the time sensitivity of this assay, standards were prepared alongside every set of test samples, and processed at the same time. Test samples were diluted where necessary to bring them within the absorbance range of the standard curve (A460 approximately 0.045 – 1.080). The whole experiment was repeated three times, and the stoichiometry of chloride release calculated for each experiment as the number of moles Cl- produced per mole of chlorinated substrate degraded. The stoichiometry values from three independent experiments were averaged and statistical analysis were carried out using GraphPad Prism software using the one-way ANOVA (and nonparametric) test.
- + QiaQuick DNA Purification Kit
- Also consult the instruction booklet that comes with the Qiagen kit – the protocol below only gives the bare essentials required. This protocol below is good for restriction fragments, plasmids, and PCR products. It is NOT good for genomic or chromosomal DNA, which is too big to stick to the column effectively. Use the FastPrep reagents or CTAB-phenol type prep instead for genomic DNA.
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- Mix your DNA sample with the appropriate buffer, in the appropriate ratio: - DNA < 4 kb: Mix 1 vol sample with 3 vol of QG buffer - DNA > 4 kb: Mix 1 vol sample with 3 vol of QG buffer + 1 vol isopropanol. - PCR products: Mix 1 vol sample with 5 vol PB buffer.
- Load the mixture onto a Qiaquick spin column (purple) and spin 30 sec. Discard the flow-through, and replace spin column in the catch tube. The spin column will hold a max. of 800μl sample and has a max. binding capacity of approx 10 μg DNA. You can wash through multiple 800 μl aliquots of DNA+QG if you have a lot of sample, so long as the total amount of DNA added doesn’t exceed approx 10 μg per column.
- Add 750 μl of buffer PE to the column, allow to sit for ~2 min, then spin 30 sec, discard flow-through, replace spin column in catch tube.
- Spin again for 30 sec to remove all traces of PE from the column. Discard both the flow-through and catch tube, and transfer spin column onto a clean Kimwipe. Leave the column lid open. Transfer Kimwipe to 50°C incubator box, and allow to dry for 10 min.
- Transfer spin column to a sterile 1.5 ml Eppi tube, and add 50 μl* of EB buffer (5 mM Tris, pH 8) to the centre of the spin column – ie on the membrane, not the walls of tube. Allow to sit for 5 min. Spin 30 sec, retain Eppi tube with DNA solution in EB, discard spin column. * Can reduce this to as little as 20 μl EB to give a more concentrated DNA solution, but keep in mind you will lose approx 3-5 μl EB during the procedure.
- Item C
- Item C.1
- Item C.2
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