Invitrogen Bac to Bac Expression System

Isolating Recombinant Bacmid DNA
Introduction
After you have transformed your pFastBac construct into DH10Bac E. coli and
performed the transposition reaction, use the procedure below to purify
recombinant bacmid DNA from DH10Bac transformants. Purified bacmid DNA
is suitable for use in PCR analysis or transfection.
Materials Needed
Have the following materials on hand before beginning.
• LB medium containing 50 µg/ml kanamycin, 7 µg/ml gentamicin, and
10 µg/ml tetracycline
• Solution I (15 mM Tris-HCl, pH 8.0, 10 mM EDTA, 100 µg/ml RNase A;
filter-sterilize and store at +4°C)
• Solution II (0.2 N NaOH, 1% SDS; filter-sterilize)
• 3 M potassium acetate, pH 5.5 (autoclave and store at +4°C)
• Isopropanol
• 70% ethanol
• 1X TE Buffer, pH 8.0
DNA Isolation
Procedure
1. Using a sterile toothpick, inoculate a single, isolated bacterial colony in 2 ml of
LB medium containing 50 µg/ml kanamycin, 7 µg/ml gentamicin, and
10 µg/ml tetracycline.
2. Grow culture in a 37°C shaking incubator until cells reach stationary phase.
3. Transfer 1.5 ml of bacterial culture to a 1.5 ml microcentrifuge tube and
centrifuge at 14,000 x g for 1 minute to pellet cells.
4. Remove the supernatant by vacuum aspiration and resuspend the cell pellet
in 0.3 ml of Solution I. Gently vortex or pipet up and down to resuspend.
5. Add 0.3 ml of Solution II and gently mix. Incubate at room temperature for
5 minutes. Note: The appearance of the suspension should change from
turbid to almost translucent.
6. Slowly add 0.3 ml of 3 M potassium acetate, pH 5.5, mixing gently during
addition. A thick white precipitate of protein and E. coli genomic DNA will
form. Place the sample on ice for 5 to 10 minutes.
7. Centrifuge for 10 minutes at 14,000 x g.
8. Gently transfer the supernatant to a microcentrifuge tube containing 0.8 ml of
isopropanol. Do not transfer any white precipitate. Invert the tube a few times
to mix and place on ice for 5 to 10 minutes. Proceed directly to Step 9 or store
sample at -20°C overnight.
9. Centrifuge the sample for 15 minutes at 14,000 x g at room temperature.
10. Carefully remove the supernatant, taking care not to disturb the pellet. Add
0.5 ml of 70% ethanol. Invert the tube several times to wash the pellet.
11. Centrifuge for 5 minutes at 14,000 x g at room temperature. Repeat Steps 10
and 11, if desired.
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