Paul.xu: Created page with "1. Incubate LB/amp or Kan with bacteria and grow cells at 37°C for 4–6 h with agitation at 220 rpm.

2. Use the 3 mL culture to inoculate a 5-L Erlenmeyer flask contai..."

2012-09-11T18:43:20Z

Created page with "1. Incubate LB/amp or Kan with bacteria and grow cells at 37°C for 4–6 h with agitation at 220 rpm. 2. Use the 3 mL culture to inoculate a 5-L Erlenmeyer flask contai..."

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

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. 

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

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

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

 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. 

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

 8.  Add '''12 mL''' of buffer '''S2''' to the suspension. Close the cap and mix gently by inverting the tube 6–8 times. 

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

 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 until a homogeneous suspension containing an off-white flocculate is formed. Let the tube stand in ice for 5 min. 

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

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

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

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

 15. Elute the plasmid DNA with 15 mL of buffer N5. Collect the eluate in a clean  centrifuge tube.

 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 at 4°C. 

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

 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. 

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

 20. Determine the plasmid yield and purity using nanodrop and  1% agarose gel. 

[[Category:Protocols]]

Plasmid Nucleobind - Revision history

Paul.xu: Created page with "1. Incubate LB/amp or Kan with bacteria and grow cells at 37°C for 4–6 h with agitation at 220 rpm. 2. Use the 3 mL culture to inoculate a 5-L Erlenmeyer flask contai..."

Paul.xu: Created page with "1. Incubate LB/amp or Kan with bacteria and grow cells at 37°C for 4–6 h with agitation at 220 rpm.

2. Use the 3 mL culture to inoculate a 5-L Erlenmeyer flask contai..."