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Agarose plug DNA


Introduction

This protocol describes a phenol-chloroform based method to purify high molecular weight genomic DNA embedded and stored in a 1%, low-melting point agarose plug. We tested this protocol using both Lambda DNA that we embedded in 1%, low-melting point agarose and S. cerevisiae DNA embedded in 1%, low-melting point agarose.

Materials

  • 1 x agarose plug
  • 2 ml Eppendorf tubes
  • Incubator, water bath or equivalent (set to 70°C)
  • TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0)
  • NaCl 5 M
  • Phenol
  • Chloroform
  • Centrifuge (capable of 9400 x g)
  • HulaMixer or equivalent
  • Ethanol
  • Ammonium acetate 5 M
  • Freezer (-20°C)

Methods

  1. Transfer an agarose plug (1% low-melt point) containing ~3-6 µg of DNA, to a 2 ml Eppendorf tube and add 200-400 µl TE to cover the agarose. If the agarose is on the tube wall, briefly centrifuge the tube. Add NaCl to a final concentration of ~200 mM.

  2. Melt the agarose at 70°C. The solution will become transparent and homogeneous, which will take approximately 5 minutes.

  3. In a fume hood, add 1 volume of phenol and gently rotate in a HulaMixer for 2 hours at room temperature.

  4. Centrifuge the tube for 5 minutes at 9400 x g.

  5. In a fume hood, retain and transfer the supernatant to a new 2 ml tube and add 1x volume of chloroform.

  6. Thoroughly but gently invert to mix. We recommend ~25 inversions.

  7. Centrifuge the tube for 5 minutes at 9400 x g.

  8. In a fume hood, retain and transfer the supernatant to a new 2 ml tube and add 2.5x volumes of 100% ethanol and 1/100 volume of 5 M ammonium acetate.

  9. Invert 10 times and incubate overnight at -20°C.

  10. Centrifuge the tube for 5 minutes at 9400 x g.

  11. Discard the supernatant and retain the pellet.

  12. Add 1 ml of ice-cold 70% ethanol and invert.

  13. Centrifuge the tube for 5 minutes at 9400 x g.

  14. Repeat Steps 11-13.

  15. Discard the supernatant and retain the pellet. Allow the pellet to air-dry for 1 minute.

  16. Resuspend the pellet in 25 µl TE and incubate at room temperature for 2 hours.

Lambda DNA

Results

  • Yield: 50-80% of initial DNA amount
  • OD 260/280: 1.88
  • OD 260/230: 1.79

Lambda DNA

Sequencing performance

Libraries were prepared using the Ligation Sequencing Kit.

  • Read length profile:

Labda DNA seq

*S. cerevisiae* DNA

Results

  • Yield: 50-80% of initial DNA amount
  • OD 260/280: 2.11
  • OD 260/230: 1.85

S. cerevisiae

Sequencing performance

Libraries were prepared using the Ligation Sequencing Kit.

  • Read length profile:

S. cerevisiae seq

Change log

Version Change
v1, June 2019 Initial protocol publication

Last updated: 7/11/2023

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