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Plasmid DNA Purification 277
are present in the elution fractions. With an appropriate gel analysis system
and via comparison to DNA markers, the concentration of pDNA in the elution
fractions can also be calculated via densitometry studies.
2. Materials
2.1. Biomolecules
The target pDNA, named pTS, is a pUC19 plasmid that has had a zinc
finger binding domain inserted into the SmaI site. Hence, the pTS plasmid is
a molecule of dsDNA 2715 base pairs in size. The pUC19 plasmid (accession
number L09137) has historically been used for general cloning (12) and
has ampicillin resistance as its method of selection. DNA sequencing of
pTS confirmed that the zinc finger binding domain sequence was present.
The plasmid molecule has a molecular weight of approximately 1800 kDa.
The pTS plasmid was produced by Dr. David Palfrey at the Department of
Pharmaceutical Sciences, Aston University (UK), and was kindly supplied by
Dr. Anna Hine. The other biomolecules used in this work (pM6, GST-ZnF and
glutathione) are described in Chapter 9.
2.2. Buffers and Reagents
Where required, use 1 M HCl or 1 M NaOH to adjust the buffer pH.
1. Growth media: Terrific broth containing 12 g tryptone, 24 g yeast extract,
K 2 HPO 4 12.5 g, 2.3 g KH 2 PO 4 in1Lofdeionized (DI) water, pH 7.
2. Phosphate-buffered saline (PBS): PBS is used as the equilibration and running
buffer. The buffer can be prepared by dissolving a PBS tablet in 200 ml of DI
water to yield a buffer containing 10 mM phosphate buffer, 2.7 mM potassium
chloride and 137 mM sodium chloride, pH 7.4.
3. Cell resuspension solution: 50 mM Tris–HCl, 10 mM ethylenediamine tetraacetic
acid (EDTA), pH 7.5.
4. Cell lysis solution: 0.2 M NaOH, 1% sodium dodecyl sulfate.
5. Neutralization solution: 1.32 M potassium acetate, pH 4.8.
6. Elution buffer: 20 mM reduced glutathione (≥99%, MW 307), 100 mM Tris–HCl,
pH 9 (prepare elution buffer on day to be used as glutathione should be stored at
4°C).
7. High pH adsorbent regeneration buffer: 0.1 M Tris-HCl, 0.5 M NaCl, pH 8.5.
8. Low pH adsorbent regeneration buffer: 0.1 M sodium acetate, 0.5 M sodium
chloride, pH 4.5.
9. Adsorbent storage buffer: 20% v/v ethanol (Sigma-Aldrich), 80% PBS.
10. Tris-EDTA (TE) buffer: 10 mM Tris–HCl, 1 mM EDTA, pH 7.
11. Tris-acetate-EDTA (TAE) buffer 50× stock: 242 g Tris, 57.1 ml glacial acetic
acid, 9.3 g EDTA, total volume adjusted to 1 l with DI water. Dilute the 50×
stock to 1× buffer on the day of use.