DWA LAB
Home
Lab Manual
biological reagents
Cell Free Systems
DNA
In Vivo Systems
misc methods
overexpression
recipes
DNA >

DNA ISOLATION AND PLASMID PURIFICATION

> Phenol extraction and ethanol precipitation.

   

> Large Scale Plasmid Prep for 100 ML Superbroth Cultures

   

> Plasmid Miniscreen Protocol (Boil Prep)

   

> Alkaline Lysis Miniprep

   

> Alkaline Lysis Miniprep Modified 1

   

> Alkaline Lysis Miniprep Modified 2

   

> Amplification of plasmids with chloramphenicol

   

> Single-Strand DNA Prep. Large Scale

    > Titre of Bacteriophage
  > Large Scale Bacteriophage Prep

Large Scale Plasmid Prep for 100 ML Superbroth Cultures

Get a printable version of this protocol (requires Adobe Acrobat

  1. Spin down the bacteria from a 100 ml culture in two 65 ml centrifuge bottles at 5000 rpm for 5 min. in the Hermle.  If you have more than will fit in the two bottles (don't exceed the line on the side of the tube or you will spray bacteria around inside the centrifuge) spin down what will fit, pour off the supernatant and then pour the rest in on top of the pellets and spin again.
  2. Discard the supernatant. Resuspend pellets in 4 ml each (8 ml total) by vortexing. Pool the solutions into one tube.
  3. Add 16 ml of Solution II, mix gently by rotating the centrifuge tube slowly. The bacteria should lyse and the solution become translucent.
  4. Add 12 ml of Solution III, shake to mix. A flocculent white precipitate should form. You are going for a cottage cheese look.  If you shake too hard your DNA will be contaminated.  If you don't shake enough the yield will be low.  Spin 5000 rpm for 10 min. to pellet the precipitate.
  5. Decant supernatant to fresh centrifuge bottle (65 ml) through gauze to trap any floating bits of precipitate. Add 0.6 volumes (25 ml) of isopropanol, mix and spin 8000 rpm for 10 minutes.
  6. Discard supernatant, remove as much as possible by aspiration. Dissolve pellet in 3 ml TE and transfer to a 12 ml centrifuge tube (Nalgene PSF tubes or disposable 14ml Falcon tubes). Add 3 ml cold (-20 C) 5M LiCl. Mix well and spin 5000 rpm for 10 minutes.
  7. Transfer supernatant to fresh 12 ml centrifuge tube, add an equal volume of isopropanol (7 ml or fill tube - don't overfill it), mix well and spin 5000 rpm for 10 minutes.
  8. Aspirate the supernatant and leave to air dry.
  9. Dissolve the pellet in 300 ul of TE + 20 ug/ml RNase A; transfer to a microfuge tube, incubate at room temperature for 15 minutes.
  10. Add an equal volume of 26% PEG in 1.6M NaCl, put on ice for 5 min, mix well and spin 15 minutes at 4C in microfuge to pellet DNA. Wash the pellet with 70% ethanol, speed vac dry before resuspending in TE. This will remove a lot of the excess PEG, making the phenol extractions much more bearable!
  11. Dissolve pellet in 400 ul of TE. (Be sure that the pellet is completely dissolved before proceeding or your DNA yield may be considerably reduced.) Extract once with an equal volume of chloroform, twice with an equal volume of buffered phenol, and once more with an equal volume of chloroform. Add ammonium acetate to 0.5M plus two volumes of 100% ethanol. Mix by gently inverting the tube several times; you should be able to see the DNA coming out of solution. Let stand for 10 minutes at room temperature, spin 5 minutes in microfuge to pellet the plasmid DNA. PHENOL IS AN ORGANIC ACID. CONTACT WITH SKIN WILL RESULT IN SEVERE BURNS. WEAR GLOVES, LAB COAT, EYE PROTECTION, AND WORK IN THE FUMEHOOD.

    CHLOROFORM IS A POISON, WEAR GLOVES, LAB COAT AND EYE PROTECTION. READ MSDS SHEETS FOR THESE MATERIALS BEFORE HANDLING.

    ETHANOL IS HIGHLY FLAMMABLE; EXERCISE CAUTION WHEN USING NEAR AN OPEN FLAME

  12. Aspirate supernatant, wash pellet with 200 ul of 70% ethanol, vortex and spin to repellet DNA. Aspirate supernatant being careful not to suck up the pellet! and dry pellet under vacuum.
  13. Dissolve DNA pellet in 50 to 100 ul of TE, depending on the size of the pellet. Read OD260 of a 1:100 dilution to quantify (1 OD unit = 50 ug/ml of dsDNA). Add TE to adjust the DNA concentration to 1 mg/ml. Store at -20 C.

    Note: when using the spectrophotometer, be sure to allow the UV lamp to warm up for at least 15 min before quanitifying your DNA. Failure to do so will result in incorrect DNA concentration.

  14. Use 0.5 - 1 ug of DNA for a restriction digest to map plasmid.
    Solution I (Plasmid Prep) Glucose Buffer
      100mL 500mL
    0.05M glucose (m.w. 180.16) 0.9g 4.504g
    0.025M Tris (pH 8.0) 1.25mL 6.25mL of (2M stock) 0.01M EDTA
      2 mL 10mL of (0.5M stock)

    pH 8.0 (should be) adjust if not

    SOLUTION II (MAKE FRESH) (PLASMID PREP)
    1% SDS 5mL of 20% SDS stock
    0.2N NaOH 5mL of 4N NaOH stock
      make up to 100mL with dH2O

    Note: put some water in container first as NaOH + SDS will ppt!

    SOLUTION III (PLASMID PREP)
      1000mL 500mL 250mL
    3.0M KoAC 294g 147g 73.5g
    2.0M HoAC 115mL 57.5mL 28.75mL
    Make up to required volume with dH2O

    pH 4.8

    Keep at -20 C this solution goes off - I don't know why!

PEG IN NaCl (100 mls)
13% PEG 8000 13 g
1.6 M NaCl 40 mL of 4 M NaCl

Make up to 100 ml with d H2O