Organic extraction of DNA

/Organic extraction of DNA
Organic extraction of DNA 2018-02-24T05:03:47+00:00

Organic extraction of DNA

Organic extraction of DNA

Introduction

Agarose that have been modified by hydroxyethylation, a substitution that reduces the number of intrastrand hydrogen bonds, melts and sets at lower temperatures than standard agarose. These properties from the basis of techniques to recover and manipulate DNA fragments in gels. In current protocol, DNA is electrophoresed on low melting temperature agarose gel, located by staining with Ethidium bromide and UV light illumination and then recovered by melting the agarose and extracting with phenol: chloroform.

Requirements

Reagents

  1. 0.7% low melting low EEO agarose
  2. Tris Cl (20mM)
  3. EDTA (1mM)
  4. PCI
  5. Sodium acetate (3M)
  6. Ethanol
  7. Distilled water
  8. MCT
  9. Head mask

Procedure

  1. DNA of interest was excised from the gel with a clean sharp scalpel.
  2. The gel slice was weighed and 1.5 volume of 20mM Tris Cl and 1mM EDTA (TE buffer) was added.
  3. The slice was incubated for 10minutes at 65ºC.
  4. The molten gel was cooled at room temperature. To this equal volume of phenol: chloroform: isoamyl was added, vortexed and spinned at 10,000rpm at RT for 5minutes.
  5. The aqueous layer was separated.
  6. To the aqueous layer 1/10th volume of sodium acetate and 2.5volume of ethanol was added.
  7. Keep the MCT at -20ºC overnight.
  8. The tube was spinned at 10,000rpm, 4ºC for 10minutes.
  9. Supernatant was discarded and the pellet was dissolved in 10µl of nuclease free water.
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