2.3 Isolation
of Bacmid DNA
and Buffer Preparation
This part of the chapter describes the materials for the isolation of
the bacmid DNA and the materials for the required buffers.
2.3.1 Materials for Buffer
Preparation
1. Tris hydrochloride buffer (Tris–HCl, e.g., Thermo Fisher Scientific, Waltham, MA, USA).
2. Ethylenediaminetetraacetic acid (EDTA, e.g., VWR, Radnor,
Pennsylvania, USA).
3. RNase A (e.g., Omega Bio-tek, Norcross, GA, USA).
4. Sodium hydroxide (NaOH, e.g., Merck, Darmstadt,
Germany).
5. Sodium dodecyl sulfate (SDS, pure Ph. Eur., AppliChem,
Darmstadt, Germany).
6. Potassium acetate (e.g., Carl Roth, Karlsruhe, Germany).
7. Distilled water, cell-culture grade (e.g., Milli-Q Academic,
Millipore, Burlington, Massachusetts, USA).
8. Sterile single-use pipette tips (e.g., serological pipettes, Sarstedt, Nu ¨mbrecht, Germany).
9. Pipette holder/rubber bulb (e.g., pipetus, Hirschmann Laborger€ ate, Eberstadt, Germany).
10. 100 mL glass bottle (e.g., Glasger€ atebau Ochs, Bovenden,
Germany).
11. Measuring cylinder (e.g., Glasger€ atebau Ochs, Bovenden,
Germany).
12. pH meter (e.g., FiveEasy™ FE20, Mettler Toledo, Columbus,
Ohio, USA).
2.3.2 Materials
for Isolation
of Bacmid DNA
1. Positive clones (clones with insert) (see Note 5).
2. Liquid LB medium for the dilution of the cell suspension (e.g.,
granulated LB medium, Carl Roth, Karlsruhe, Germany, prepare according to the manufacturer’s instructions).
3. Kanamycin and tetracycline in corresponding working concentrations (see Note 4).
4. Cultivation tubes (e.g., Sarstedt, Nu ¨mbrecht, Germany).
5. Resuspension buffer (containing Tris-HCl and EDTA).
6. Lysis buffer (containing NaOH and SDS).
7. Precipitate buffer (containing potassium acetate).
8. Absolute isopropanol (e.g., Merck, Darmstadt, Germany).
9. 70% (v/v) ethanol (e.g., Merck, Darmstadt, Germany).
10. Distilled water, cell-culture grade (e.g., Milli-Q Academic,
Millipore, Burlington, Massachusetts, USA).
Antigen Production in Insect Cells
99