Griffitts:Midiprep

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Contents

Materials

  • Buffer RES (contains RNase—store at 4°C)
  • Buffer LYS (in Nucleobond kit)
  • Buffer NEU (in Nucleobond kit)
  • Buffer EQU (in Nucleobond kit)
  • Buffer ELU (in Nucleobond kit), warmed to 60°C
  • Isopropanol
  • 70% ethanol
  • T5E0.5
  • Nucleobond columns (one per sample)
  • 40-mL centrifuge tubes, autoclaved (four per sample)
  • 1.7-mL microcentrifuge tubes (five per sample)
  • 50-mL Falcon tubes (two per sample)

Procedure

NOTE: Do NOT use pipetting for resuspension—it can cause shearing of the genomic DNA.

Lysis

  1. Label all tubes
    • Label the microcentrifuge tubes ELU1 to ELU5
    • Draw a line at the 1-mL mark on all microtcentrifuge tubes
  2. Grow S. meliloti cultures in 200-mL LB overnight to saturation
  3. Centrifuge four 40-mL aliquots of each culture at 8,000 rpm for 8 minutes at 4°C
    • Use an SS-34 rotor
  4. Discard supernatant
  5. Thoroughly resuspend each pellet in 5 mL RES Buffer (in fridge)
  6. Combine samples into two tubes (each should now have 10 mL)
    • Be careful not to mix samples from different cultures!
  7. Add 10 mL Buffer LYS
  8. Invert 4 times
  9. Incubate at room temperature for 3 minutes
  10. Add 10 mL Buffer NEU
  11. Invert 8 times
  12. Incubate at room temperature for 3 minutes
  13. Centrifuge at 11,000 rpm for 5 minutes at 4°C

Midiprep Column

  1. Suspend a Nucleobond column over a 50-mL Falcon tube using a white tip holder
    • It may be necessary to put tape on the column so that it doesn't slide down too far
  2. Equilibrate the column with 12 mL Buffer EQU and allow it to empty by gravity flow
  3. Dump the supernatant from each sample (two tubes with 30 mL each) into a column and allow it to pass through the filter by gravity flow
    • As the 50-mL Falcon tube fills up, transfer the column to a new one
  4. Wash the column filter with 5 mL of Buffer EQU and allow it to move through by gravity flow
    • Apply to the sides of the filter, not the bottom
  5. Remove the column filter and discard
  6. Wash the column with 8 mL of Buffer EQU and allow it to move through by gravity flow
  7. Warm the Buffer ELU to 60°C (to increase yields) in one of the following ways:
    • Microwave for ~5 seconds
    • Set in the 60°C water bath for ~5 minutes
  8. Elute with 5.2 mL of 60°C Buffer ELU and allow it to move through by gravity flow
  9. Collect eluent in 5 microcentrifuge tubes (1 mL per tube)
  10. Discard tube ELU1
  11. To tubes ELU2 through ELU5 add 700 μL of isopropanol (this precipitates the DNA)
  12. Incubate at –20°C for 30 minutes
  13. Centrifuge at maximum speed for 10 minutes
    • Do this in the cold room
  14. Remove most of the isopropanol
  15. Centrifuge at maximum speed for 1 minute
  16. Remove the rest of the isopropanol with a P-200
  17. Drizzle 150 μL cold 75% ethanol down the back of each tube
    • Do not pipette up and down or vortex!
  18. Centrifuge at maximum speed for 5 minutes
    • This can be done at room temperature
  19. Remove all of the ethanol with a P-200
    • Be cautious—the pellets may be very loose
  20. Add 50 μL of T5E0.5
    • Drizzle the TE buffer twice down the back of the tube
    • Do not pipette up and down or vortex!
  21. To dissolve the DNA, incubate at 60°C and flick once every minute for 15 minutes

Analysis

  1. Analyze by gel electrophoresis
  2. Run 5 μL on a 1% gel for 15 minutes at 130 V


NOTE: This worked very well for low/medium copy plasmid pJG442.


Adapted from the Nucleobond Xtra Midi handbook. (Clontech cat# 740410.01)

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