The previous protocol works well for the majority of bacmids in our labs. However, the alternative protocol given here produces higher quality bacmid, and gives greater numbers of viruses after transfection. This alternative approach is supported by a Life Technologies technical paper.

Inoculation

Inoculate a single, white bacterial colony into 2 ml LB containing kanamycin at 50 µg/ml and gentamycin 7µg/ml (20 ml universal or 50ml falcon tube). Place the broth culture in a shaking incubator or water bath at 250 rpm, 37°C for a minimum of 16 hours (overnight is fine).

Isolation of recombinant bacmid DNA using the Life Technologies CONCERT High Purity Plasmid Purification System

1. Before beginning: verify that no precipitate has formed in Cell Lysis solution (E2). If the solution is too cold, the SDS will precipitate out of the solution. Note: Remember to add RNase A to cell Suspension Buffer (E1).
2. Column Equilibration: Apply 2 ml of Equilibration Buffer (E4) (600mM NaCl, 100mM sodium acetate (pH 5.0), 0.15% Triton X-100) to the column. Allow the solution in the column to drain by gravity flow.
3. Cell Harvesting: Pellet 1.5ml of an overnight culture. Thoroughly remove all medium.
4. Cell suspension: Add 0.4ml of cell suspension Buffer (E1) 50mM Tris-HCL(pH 8.0), 10mM EDTA, containing RNase A 0.2 mg/ml) to the pellet and suspend cells until homogenous.
5. Cell Lysis: Add 0.4ml of Cell lysis solution (E2) (200mM NaOH, 1% SDS). Mix gently by inverting the capped tube five times. Do not vortex. Incubate at room temperature for 5 min.
6. Neutralization: Add 0.4ml of Neutralisation Buffer (E3) (3.1M potassium acetate (pH5.5) and mix immediately by inverting the tube 5 times. Do not vortex. Centrifuge the mixture at top speed in a microfuge at room temperature for 10min. Do not centrifuge at 4°C.
7. Column Loading: Pipette the supernatant from step 6 onto the equilibrated column. Allow the solution in the column to drain by gravity flow. Discard flow-through.
8. Column wash: Wash the column two times with 2.5ml of wash buffer (E5)(800 mM NaCl, 100mM Sodium acetate (pH5.0). Allow the solution in the column to drain by gravity flow after each wash. Discard flow-through.
9. Plasmid DNA Elution: Elute the DNA by adding 0.9ml of Elution Buffer (E6) (1.25 M NaCl, 100mM Tris-HCl (pH8.5). Allow the solution in the column to drain by gravity flow. Do not force out remaining solution.
10. Plasmid DNA Precipitation: Add 0.63ml of isopropanol to the eluate. Mix and place on ice for 10min. Centrifuge the mixture at top speed in a microfuge at RT for 20min. Carefully discard supernatant. Wash the plasmid DNA pellet with 1 ml of ice cold 70% ethanol and centrifuge for 5 min. Carefully and fully pipette off the ethanol wash. Air dry the pellet for 10 min.
11. Purified DNA: Dissolve the pelleted DNA in 40µl of Ultra Filtered water. Allow DNA to dissolve for at least 10min on ice. To avoid DNA shearing, pipette DNA only 1-2 times during resuspension. Note- Bacmid DNA can be stored at -20°C,but avoid repeated freeze/thawing.