Biology Protocols

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Large Scale Plasmid Preparation

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  • Grow 500ml-1litre cultures to saturation (the smaller volume results in a more efficient extraction).
  • Spin the cells down in the RC-3B (3K; 20m; 4°C)
  • Resuspend the cells in 30ml of GTE buffer:

     50mM glucose
     25mM Tris HCl pH 8.0
     10mM EDTA

      and transfer to 250ml tubes. Cool on ice.

  • Add 60ml of ice-cold 0.2M NaOH/1% SDS, mix well by rolling the tubes and leave on ice for 5m exactly.
  • Add 30ml of 3M KHAc, pH 4.8. Mix well by rolling the tubes and leave on ice for 15m. (This timing is not critical.)
  • Spin the tubes in the RC-5B (GSA rotor) (10K; 25-30m; 4o). Filter the supernatant through two layers of cheese cloth into fresh 250ml tubes.
  • If the supernatant is not clear, repeat the previous step. This is very important.
  • Precipitate the nucleic acids by adding 75ml of isopropanol (~0.6vol) and centrifuge immediately in the RC-5B (10K; 10m; R/T).
  • Pour off the supernatant and discard. Dissolve the pellet in 10ml of 10x TE and transfer to 50ml Oakridge tubes.
    Add 50μl RNAase A (20mg/ml) and leave at 37° for 30m.
    Add 1ml 3M NaCl (or NaAc).
  • Add 10ml of buffered phenol, cap the tube and shake well. Centrifuge in the RC-3B (five-place, round-bottomed adapters) (4K; 15m; R/T) or in the RC-5B (10K; 5-10m; R/T).
  • Remove the upper phase into fresh tubes and repeat (9) once or twice, until no interphase is seen after centrifugation.
  • Precipitate the nucleic acids with 20ml of ice-cold ethanol, mix well and leave at -20°C for 30m.
  • Spin the tubes in the RC-5B (10K; 15m+;4°C). Pour off the supernatant and discard, leaving the tubes upside down to drain for a minute.
  • To remove the bulk of RNA by differential precipitation, redissolve the pellets in 10ml of 0.3M NaAc ~pH 7 at R/T. Add 5.6-5.8ml isopropanol at R/T dropwise in a whirlymixer. Leave at R/T for 10m then centrifuge in the RC-5B (10K; 15m; R/T).
  • Retain the supernatant in case the DNA has not precipitated.
  • Wash the pellet with 10ml of 0.3M NaAC/ isopropanol, (1:0.6 v/v) and spin again for 5m.
    Discard the supernatant, add 10ml chilled ethanol and centrifuge again, setting the temperature to 4°C.
  • Discard the supernatant and leave the tubes to drain for a few minutes. Keep an eye on medium-sized pellets as they sometimes slide down the side of the tube.

Notes

The DNA may be further purified by PEG purification or by CsCl gradient.If you are using CsCl centrifugation the isopropanol/NaAc and ethanol washes may be omitted.
The RNAase A step may be omitted together with the differential precipitation as the RNA will pellet on the CsCl gradient. However it is necessary to be quick or the RNA will degrade and contaminate the DNA.

 

 


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