Editing Protein Production using Baculovirus (section)

== Bacmid DNA miniprep  ==

<br>1. Grow a single colony in 5 mL of LB containing 50 μg/mL kanamycin, 7 μg/mL gentamicin, 10 μg/mL tetracycline overnight. It’s best to start these cultures in the<br>morning, since the cells grow very slowly. 

2. Make a glycerol stock by mixing 500 μL of the cells with 500 μL of 80% glycerol. Store in the -80 °C freezer.<br>3. Transfer the remaining culture to a 2-mL microcentrifuge tube and spin at maximum speed for 1 min.<br>4. Remove all of the supernatant using the vacuum aspirator.<br>5. Resuspend the cells in 300 μL of Qiagen Buffer P1. Do not use P1 containing the blue lysis indicator dye.<br>6. Add 300 μL of Qiagen Buffer P2, then incubate at room temperature for 5 min.<br>7. Slowly add 300 μL of Qiagen Buffer N3, mixing gently during addition. A thick white precipitate of protein and genomic DNA will form. Place the sample on ice for 5-10 min.<br>8. Centrifuge for 10 min at maximum speed. During this step, label another tube and add 800 μL isopropanol to it.<br>9. Gently transfer the supernatant into the tube containing the isopropanol. Avoid transferring any of the white precipitate. Mix gently by inverting the tube a few times and place on ice for 5-10 min. Note: The sample can be stored at -20 °C overnight (or over a weekend) to get higher yield.<br>10. Centrifuge for 15 min at maximum speed.<br>11. Remove the supernatant and add 500 μL of 70% ethanol. Invert the tube several times to wash the pellet. Centrifuge for 10 min at maximum speed. Repeat the wash step.<br>12. Inside biosafety hood, carefully remove all of the supernatant.<br>13. Do not dry the pellet. Resuspend the DNA in 50~100 μL of TE buffer. <br>14. Store DNA at 4 °C, until it is used for transfection. After transfection transfer plasmid to -20 °C freezer for long-term storage. Avoid repeated freeze-thaw cycles.<br><br>