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Methods:Genomic DNA prep

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Contents

Purpose

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Materials Needed

Protocol

Adapted from Syn & Swarup (2000)[1].

  1. Centrifuge 3mL of an overnight culture grown in LB to pellet cells.
  2. Resuspend in 0.75mL 1% NaCl. Centrifuge.
  3. Resuspend pellet in 0.75mL TES (10mM Tris-HCl, 10mM EDTA, pH 8.0, 2% SDS)
  4. Incubate at 75oC for 15 minutes. Vortex.
  5. Add 1ml phenol:chloroform (3:1 v/r). Centrifuge.
  6. Pipet off the aqueous layer (top) into a fresh tube.
  7. Add 1mL chloroform. Centrifuge.
  8. Pipet off the aqueous layer (top) into a fresh tube.
  9. Add 100μL 3M NaOAc (pH 5.2) & 1mL isopropanol.
  10. Invert tube & centrifuge.
  11. Add 500μL absolute ethanol & spool out DNA into fresh tube.
  12. Resuspend DNA (spooled) in 150uL TE (10mM Tris-HCl, 2mM EDTA, pH 8.0) with 1μL 50ug/mL RNase.

Usage Examples

User notes

    • Do NOT get the interface or the bottom layer during phenol:chloroform extraction!

Related GO Terms

References

  1. Syn, CK & Swarup, S (2000) A scalable protocol for the isolation of large-sized genomic DNA within an hour from several bacteria. Anal. Biochem. 278 86-90 PubMed EcoliWiki page