Editing Plasmid Nucleobind

1. Incubate LB/amp or Kan with bacteria and grow cells at 37°C for 4–6 h with agitation at 220 rpm.<br><br>

2. Use the 3 mL culture to inoculate a 5-L Erlenmeyer flask containing 1 L of LB broth with 100 g/mL ampicillin or 50 g/mL kanamycin depending on the plasmid.<br>

<br>3. Incubate 12–16 h at 37°C with agitation at 220 rpm.<br>

<br>4. Transfer the culture to two 500-mL centrifuge bottles.<br>

<br>5. Centrifuge at 5,000 × g for 20 min at 4°C and decant the medium<br>

<br>6. Resuspend each cell pellet in '''12 '''mL of buffer '''S1''' from the NucleoBond AX 500 kit (Macherey-Nagel). Completely resuspend the cells by pipetting with a 10-mL pipette.<br>

<br>7. Transfer the resuspended cells into a 50-mL centrifuge tube.<br>

<br>8.&nbsp; Add '''12 mL''' of buffer '''S2''' to the suspension. Close the cap and mix gently by inverting the tube 6–8 times.<br>

<br>9. Incubate the mixture at room temperature (20–25°C) for 2–3 min. Do not vortex, as this will release chromosomal DNA<br>from the cellular debris.<br>

<br>10. Add '''12 mL''' of prechilled (4°C) buffer '''S3''' to the suspension. Close the cap and mix gently by inverting the tube 6–8 times<br>until a homogeneous suspension containing an off-white flocculate is formed. Let the tube stand in ice for 5 min.<br>

<br>11. Centrifuge the suspension at 5,000 × g for 30 min at 4°C. Filter supernatant with coffe filter paper.<br>

<br>12. Attach one NucleoBond AX 500 column to a support stand and equilibrate the column with '''6 mL of buffer N2'''. Allow the<br>column to empty by gravity flow and discard the flow-through.<br>

13. Load the cleared lysate supernatant onto the NucleoBond column. Allow the column to empty by gravity flow.

<br>14. Wash the column twice with '''32 mL''' of buffer '''N3'''. Collect the flow-through in a beaker and then discard.

<br>15. Elute the plasmid DNA with 15 mL of buffer N5. Collect the eluate in a clean&nbsp; centrifuge tube.

<br>16. Add 11 mL of isopropanol at room temperature to precipitate the plasmid DNA. Mix well and centrifuge at 4,000 × g for 1 h<br>at 4°C.<br>

17. In a laminar flow hood, carefully pour the supernatant into a waste container.

<br>18. Rinse the pellet with 2 mL of 70% ethanol and carefully decant the 70% ethanol. Allow the pellet to air dry in the hood at room temperature.<br>

19. To the pellet add 0.7 mL of sterile TE to dissolve DNA.

<br>20. Determine the plasmid yield and purity using nanodrop and&nbsp; 1% agarose gel.<br><br><br>

[[Category:Protocols]]