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œœœœœ@Giant plasmid mini-prep@œœœœœ



For most purposes, such as sequencing, T7 transcription, subcloning, etc, the time & expense of CsCl:ethidium bromide purification of plasmid DNAs is not required. This procedure is essentially a scaled-up miniprep procedure that, because of the extensive organic extractions, is of sufficient purity for most purposes.
If the DNA doesn't cut when you're done, or you need to get rid of the last of the RNA, you can purify this DNA using CsCl:EtBr, as usual (see CsCl purification of plasmid DNA).


  1. Materials

    1. Sucrose buffer
      25% SUCROSE (w/v)
      50 mM Tris-HCl pH 8.0

    2. LYSOZYME SOLUTION
      416 ul 1M Tris-HCl (pH 8.0)
      41.6 mg Lysozyme
      1.6 ml 0.5 M EDTA (pH 8.0)
      3 ml ddH2O
      5 ml

    3. TRITON LYSING SOLUTION :
      2% Triton
      10 mM Tris-HCl pH8.0
      62.5 mM EDTA pH 8.0

  2. Methods

    1. Start a 10 ml culture of desired strain in a drugged medium and allow to grow for 4-6 hours (mid to late log phase).
    2. From this inoculate 500 ml of drugged media with 2 ml of cells and allow to grow at 37‘C over night.
    3. Transfer the culture to two 250 ml centrifuge bottles and cool on ice for 15 min.
    4. Spin cells down in GSA rotor at 7.5K, 4‘C, for 10 min.
    5. Pour off the supernatant, drain the bottles, and wipe the walls of the bottle with a Kimwipe.
    6. Resuspend the cells (both pellets) in 5 ml Sucrose Buffer (the suspension will be almost opaque) and transfer to a 25ml 60Ti tube.
    7. Add 5 ml of 10 mg/ml Lysozyme in TE Buffer and mix the tube gently.
    8. Leave on ice for 15 min.
    9. Add 10 ml of Triton lysing solution to the cells, seal the tube with Parafilm and mix by inverting the tube gently.
    10. Leave on ice for 20 min.
    11. Spin the tube at 35K, 30 minutes at 4C in the 60Ti rotor (be sure its precooled).
    12. Pour the supernatant into a 35ml Corex tube and seal with Parafilm.
    13. Heat the tube to 60‘C for 5 min. to ppt. the proteins. The solution should become cloudy.
    14. Spin tube 10 min. 4‘C at 10K.
    15. Transfer the supernatant to two 30 ml Corex tubes and extract each with 10 ml TE saturated phenol (pH 8.0). (The tubes should be sealed with a silicone #3 stopper and shaken for 30 sec. Since this is an organic extraction pressure may build up in the tube, so remember to burp the tube during the extraction and be careful when opening the tube. Spin the tubes in the table top clinical centrifuge for two min. to separate the two phases. Always wear gloves when working with phenol and/or chloroform.)
    16. Repeat the extraction two times with 1:1, phenol:chloroform, and then with 10ml of chloroform.
    17. Divide into two corex tubes & isopropanol ppt. the DNA with 4ml 7.5M NH4OAc and 28 ml isopropanol in each sample. Chill the tubes in ice for 15 min.
    18. Pellet the DNA by centrifugation at 10K, 4‘C, for 20 min.
    19. Resuspend the pellets in a total of 5 ml of 1X TE.
    20. Transfer to a 15 ml screw cap dispo tube and add 50 ul of 10 mg/ml RNaseA. Seal the tube with its cap and secure the cap with Parafilm.
    21. Incubate the tube for 1 hour at 37‘C.
    22. Add 5 ml of saturated phenol to the tube and shake vigorously for 30 sec. and spin as above (15).
    23. Transfer the aqueous phase to a 15 ml Corex tube and extract with 5 ml 1:1 , phenol : chloroform. Then extract with 5 ml of chloroform.
    24. Isopropanol ppt. with 0.4 vol 7.5M NH40Ac and 2 vol. isopropanol.
    25. Spin out the DNA and rinse the pellet with 70% ethanol.
    26. Dry the tube walls with a Kimwipe and decicate the pellet lightly.
    27. Resuspend the pellet in 1 ml 50 mM tris-HCl (pH 8.0). Quantitate the DNA by diluting 4 ul of your sample into 1 ml of H2O and reading the absorbance at 260 nm and 280 nm. Figure an A260 of 1.0 = 40 ug of DNA.


PaceLabs
Dirk Hunt, personal communication

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