Template:Team:SydneyUni Australia/ProtocolHTML

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Revision as of 12:46, 14 September 2013

  • + Chloride Assay
    • 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.

    • • 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.

    • • 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.]


  • + 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.
      1. 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.
      2. 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.
      3. 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.
      4. 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.
      5. 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.
        6. * 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.

  • + Plasmid Mini Prep (100 mL)
      1. Pellet 100 ml culture in 2 x 50 ml Falcon tubes. Resuspend cells in 4 ml TE buffer, combine resuspended pellets in one tube.
      2. Add 8 ml lysis solution (SDS-OH), mix well by inversion and shaking(~10 sec). Should go viscous. Leave at room temp for 15 min.
      3. Add 6 ml ice-cold precipitation solution (K.Ac). Shake briefly – essential that K.Ac is thoroughly mixed in. Viscosity should disappear, and white precipitate appears. Keep on ice 15 min.
      4. Spin at top speed (4000 rpm) in cold Centaur centrifuge for 15 min. Recover tube immediately and handle gently (pellet is soft and easily resuspended). Pour supernatant into new tube. Try to avoid the white junk, but don’t worry if little bits of it get transferred.
      5. Add an equal volume isopropanol (~15 ml), mix well ice 15 min.
      6. Spin at top speed (4000 rpm) in Centaur centrifuge (doesn’t need to be cold) for 15 min., pour off supernatant, keep pellet.
      7. Add 10 ml 70% ethanol to pellet, resuspend by brief vortexing, leave for 5 min at room temp. Spin 15 min in Centaur centrifuge (doesn’t need to be cold). Pour off supernatant again.
      8. Drain off excess supernatant by gentle tapping on paper towel, then heat at 50°C for approx 10 min to remove ethanol and isopropanol.
      9. Redissolve pellet in 2 ml TE with mixing (tapping tube ~ 1 min , don’t vortex too much from this point onward). Can heat if necessary (eg. 50°C, 10-30 min). Note that plasmid DNA is much more soluble than chromosomal DNA, and dissolves preferentially. Solution should look slightly viscous (traces of chrom DNA still present).
      10. Split prep into 2 x 1 ml in Eppi tubes. Extract each tube with phenol: chloroform: isoamyl (PCI), as follows. Suck up 500 µl PCI from under the aqueous layer in the reagent bottle, transfer to Eppi tube. Vortex for ~5-10 sec until a uniform milky white emulsion is obtained. Centrifuge 5 min. Transfer top phase (aqueous) to a new Eppi tube. Discard bottom phase (PCI) into phenol waste. Avoid the white junk at the interface between phases.
      11. Repeat solvent extractions using 500 µl chloroform:isoamyl (CI, µl) per tube, as described for PCI above. After mixing & centrifugation, keep top aqueous phase, discard bottom CI phase into waste.
      12. Split DNA prep into 4 equally sized aliquots (~400 µl each) Precipitate DNA by adding 1/10-volume 3M Na-acetate (~40 µl) to each tube, and then add 2 volumes cold ethanol (~1 ml). Incubate >2 hr at -20°C (overnight is fine)
      13. Spin for 10 min, drain off supernatant, rinse pellet with 70% ethanol (as above, but using 500 µl 70% EtOH), drain excess EtOH off, then dry 10 min at 50°C.
      14. Redissolve plasmid in 100 µl EB. Expected yields range from approx 10 µg plasmid per ml culture (eg pUC/pGEM) down to 0.2 µg plasmid per ml culture (eg. RSF1010). Final expected DNA conc. may range from approx 10-500 ng/µl.


        15. Solutions: (see Sambrook Appendix 1)

        • TE (solution I): 10 mM Tris, 10 mM EDTA, pH 8. Autoclaved.
        • Lysis sol’n (solution II): 0.2 M NaOH, 1% SDS. Prepare fresh from separate stocks (NaOH – 2 M, Autoclaved ; SDS – 10%)
        • Precipitation sol’n (solution III): 3 M potassium, 5 M acetate, pH 4.8. Autoclaved.
        • Na-acetate: 3M, pH 4.8 (adjust with conc. acetic acid), autoclaved.
        • EB (Elution buffer): 5 mM Tris, pH 8. Autoclaved.

        NOTE: RNAse can be added to TE buffer at the start, or to the EB/TE at the end. RNA doesn’t interfere with most things, but can make gels look messy and obscure small DNA bands. Add RNase from conc., boiled stock (10 mg/ml) to final conc. of ~100 µg/ml.

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