Home Protocols DNA Purification from Agarose Gels using Beta Agarase I (NEB #M0392)

DNA Purification from Agarose Gels using Beta Agarase I (NEB #M0392)

Overview

β-Agarase I can be used to purify both large (>50 kb) and small (<50 kb) fragments of DNA from agarose gels, and the resulting carbohydrates can be removed if necessary.

Introduction

Large DNA Fragments:Fragments larger than 50 kb require delicate handling to avoid mechanical shearing, so we advise that subsequent manipulation be carried out in the digested agarose or that a drop dialysis step be introduced to remove carbohydrates and β-Agarase I (MW 30,000).

Small DNA Fragments:Alcohol precipitation of DNA (carbohydrates remain in solution).

Protocol

Agarose Digestion:

This procedure will digest up to 200 µl of 1% low melting point agarose. For larger volumes, adjust enzyme accordingly.

  1. Equilibrate the DNA-containing low melting point agarose (SeaPlaque GTG or NuSieve GTG) by washing the solid gel slice twice with 2 volumes of 1X b-Agarase I Buffer on ice for 30 minutes each.*
  2.  Remove the remaining buffer and melt the agarose by incubation at 65°C for 10 minutes.
  3.  Cool to 42°C and incubate the molten agarose with 1 unit of β-Agarase I at 42°C for 1 hour. Proceed to DNA Purification.

    *As an alternative method of equilibration, add 1/10 volume of 10X β-Agarase I Reaction Buffer and melt together with the agarose. This faster equilibration method requires the amount of enzyme used to be doubled. This method is recommended when working with DNA fragments shorter than 500 base pairs because it avoids diffusion of DNA during washing.

DNA Purification:

  1. Adjust the salt concentration of the β-Agarase I treated solution for isopropanol precipitation of DNA (0.5 M NaCl, 0.3 M NaOAc, 2.5 M NH4OAc or 0.8 M LiCl).
  2. Chill on ice for 15 minutes.
  3. Centrifuge at 15,000 X g for 15 minutes to pellet any remaining undigested carbohydrates.
  4. Remove the DNA-containing supernatant. Precipitate with 2 volumes of isopropanol. To ensure quantitative yields of small quantities of DNA (< 100 ng), carrier RNA (1 µg) can be added to the solution.
  5. Mix thoroughly, chill and centrifuge at 15,000 X g for 15 minutes.
  6. Remove the supernatant, wash the pellet with cold 70% isopropanol and dry the pellet at room temperature.
  7. The pellet can be resuspended in TE or any buffer necessary for subsequent manipulation.

For a faster solution, the Monarch DNA Gel Extraction Kit (NEB #T1020) can be used to extract DNA from agarose gels.