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[Bio101] E. coli Genomic DNA Extraction

Microbiology > Microbial genetics > DNA
Author: Fanglian He
7/20/2011, 40308 views, 36 Q&A

[Abstract] This protocol uses phenol/chloroform method to purify genomic DNA without using commercial kits.

Materials and Reagents

  1. Tris base (Calbiochem-Behring)
  2. Proteinase K (Sigma-Aldrich)
  3. Phenol\chloroform (1: 1) (EM Science)
  4. 200 proof ethanol (Pharmco-AAPER)
  5. RNAase (Life Technologies, Invitrogen™)
  6. Ethanol
  7. SDS
  8. EDTA
  9. Tryptone
  10. Yeast extract
  11. NaCl
  12. LB medium (see Recipes)
  13. TE buffer (see Recipes)
  14. Lysis buffer (see Recipes)

Equipment

  1. Tabletop centrifuge (Eppendorf)
  2. 1.5 ml Eppendorf tube
  3. Incubator
  4. Gloves

Procedure

  1. Transfer 1.5 ml of the overnight E. coli culture (grown in LB medium) to a 1.5 ml Eppendorf tube and centrifuge at max speed for 1min to pellet the cells.
  2. Discard the supernatant.
    Note: Remove as much of the supernatant as you can without disturbing the cell pellet.
  3. Resuspend the cell pellet in 600 μl lysis buffer and vortex to completely resuspend cell pellet.
  4. Incubate 1 h at 37 °C.
  5. Add an equal volume of phenol/chloroform and mix well by inverting the tube until the phases are completely mixed.
    Note:
    Do not vertex the tube—it can shear the DNA.
  6. CAUTION: Phenol is a very strong acid that causes severe burns. Chloroform is a carcinogen. Wear gloves, goggles and lab coat, and keep tubes capped tightly. To be safe, work in the hood if possible.
  7. Spin at max speed for 5 min at RT (all spins are performed at RT, unless indicated otherwise). There is a white layer (protein layer) in the aqueous: phenol/chloroform interface.
  8. Carefully transfer the upper aqueous phase to a new tube by using 1 ml pipetman (to avoid sucking the interface, use 1 ml tip with wider mouth-cut 1 ml tip-mouth about ~2 mm shorter).
  9. Steps 4-6 can be repeated until the white protein layer disappears.
  10. To remove phenol, add an equal volume of chloroform to the aqueous layer. Again, mix well by inverting the tube.
  11. Spin at max speed for 5 min.
  12. Remove aqueous layer to new tube.
  13. To precipitate the DNA, add 2.5 or 3 volume of cold 200 proof ethanol (store ethanol at -20 °C freezer) and mix gently (DNA precipitation can be visible).
  14. Incubate the tube at -20 °C for 30 min or more.
  15. Spin at max speed for 15 min at 4 °C.
  16. Discard the supernatant and rinse the DNA pellet with 1 ml 70% ethanol (stored at RT).
  17. Spin at max speed for 2 min. Carefully discard the supernatant and air-dry the DNA pellet (tilt the tube a little bit on paper towel). To be faster, dry the tube at 37 °C incubator.
  18. Resuspend DNA in TE buffer.
    Note: Large amounts of RNA will be present in the DNA sample. So, for subsequent reactions, for example, to digest plasmid DNA, add 1-5 μl (1 mg ml-1) RNAase to the digestion solution to completely remove RNA. Or, add RNAase directly to lysis buffer with a final concentration of 1 mg ml-1.

Recipes

  1. LB medium
    1% tryptone
    0.5% yeast extract
    200 mM NaCl
  2. TE buffer
    10 mM Tris-Cl (pH 8.0)
    1 mM EDTA (pH 8.0)
  3. Lysis buffer (10 ml)
    9.34 ml TE buffer
    600 ul of 10% SDS
    60 μl of proteinase K (20 mg ml-1)


How to cite this protocol: (2011). E. coli Genomic DNA Extraction. Bio-protocol Bio101: e97. http://www.bio-protocol.org/e97



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