β-Agarase I can be used to purify both large (>50 kb) and small (<50 kb) fragments of DNA from gels, and the resulting carbohydrates can be removed if necessary.
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).
- 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).
- Chill on ice for 15 minutes.
- Centrifuge at 15,000 X g for 15 minutes to pellet any remaining undigested carbohydrates.
- 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.
- Mix thoroughly, chill and centrifuge at 15,000 X g for 15 minutes.
- Remove the supernatant, wash the pellet with cold 70% isopropanol and dry the pellet at room temperature.
- The pellet can be resuspended in TE or any buffer necessary for subsequent manipulation.