Isolation of genomic DNA

/Isolation of genomic DNA
Isolation of genomic DNA 2018-02-23T11:45:25+00:00

Isolation of genomic DNA

Isolation of genomic DNA

Introduction

Bacteria from a saturated liquid culture are lysed and proteins removed by digestion with proteinase K. Cell wall debris, polysaccharides, and remaining proteins are removed by selective precipitation with CTAB, and high-molecular-weight DNA is recovered from the resulting supernatant by isopropanol precipitation.

Requirements

Reagents

  1. TE buffer
  2. 10% sodium dodecyl sulfate (SDS)
  3. 20 mg/ml proteinase K (stored in small single-use aliquots at −20°C)
  4. 5 M NaCl
  5. CTAB/NaCl solution (10% CTAB in 0.7M NaCl)
  6. 24:1 chloroform/isoamyl alcohol
  7. 25:24:1 phenol/chloroform/isoamyl alcohol
  8. Isopropanol
  9. 70% ethanol

Equipments

  1. Laminar air flow
  2. Refrigerated microcentrifuge
  3. Incubator
  4. Refrigerator
  5. Vortex

Procedure

  1. Inoculate a 5-ml liquid culture with the bacterial strain of interest. Grow in conditions appropriate for that strain (i.e., appropriate medium, drug selection, temperature) until the culture is saturated. This may take several hours to several days, depending on the growth rate.
  2. Spin 1.5 ml of the culture in a microcentrifuge for 10 min, or until a compact pellet forms. Discard the supernatant.
  3. Resuspend pellet in 567 μl TE buffer by repeated pipetting. Add 30 μl of 10% SDS and 3 μl of 20 mg/ml proteinase K to give a final concentration of 100 μg/ml proteinase K in 0.5% SDS. Mix thoroughly and incubate 1 hr at 37°C.
  4. Add 100 μl of 5 M NaCl and mix thoroughly.
  5. Add 80 μl of CTAB/NaCl solution. Mix thoroughly and incubate 10 min at 65°C.
  6. Add an approximately equal volume (0.7 to 0.8 ml) of chloroform/isoamyl alcohol, mix thoroughly, and spin 10 to 12 min in a microcentrifuge.
  7. Remove aqueous, viscous supernatant to a fresh microcentrifuge tube, leaving the interface behind. Add an equal volume of chloroform:isoamyl alcohol, extract thoroughly, and spin in a microcentrifuge for 5 min.
  8. Transfer the supernatant to a fresh tube. Add 0.6 vol isopropanol to precipitate the nucleic acids (there is no need to add salt since the NaCl concentration is already high). Shake the tube back and forth until a stringy white DNA precipitate becomes clearly visible. At this point it is possible to transfer the pellet to a fresh tube containing 70% ethanol by hooking it onto the end of a micropipet that has been heat-sealed and bent in a Bunsen flame. Alternatively, the precipitate can be pelleted by spinning briefly at room temperature.
  9. Wash the DNA with 70% ethanol to remove residual CTAB and respin 5 min at room temperature to repellet it. Carefully remove the supernatant and briefly dry the pellet in a lyophilizer.
  10. Redissolve the pellet in 100 μl TE buffer.
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