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MOLECULAR BIOLOGY: WORKING WITH DNA

DOT BLOT ANALYSIS

Dot Blotting of DNA

Dot Blotting of DNA Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Contributor:	Simon Dawson at the School of Biomedical Sciences, Queen's Medical Centre
Overview Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
This protocol describes how to prepare a DNA dot blot with a dot blot apparatus and a vacuum manifold.
Procedure Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
1. Dot blots typically use nylon membranes, either Hybond-N or Zeta-Probe. 2. Wash the dot blot apparatus thoroughly with 1% SDS, then with sterile water prior to use. 3. Cut a piece of nylon membrane and Whatman 3MM filter paper to cover the required number of wells of the apparatus (see Hint #1). 4. Soak both the nylon membrane and the filter paper in 2X SSC for 5 minutes prior to use. 5. Place the Whatman paper into the dot blot apparatus with the nylon membrane placed on top. Clamp the entire structure together. 6. Dilute DNA samples to 100 μl with sterile ddH2O. 7.Boil the samples for 10 minutes, then quickly chill in an ice-water bath. 8. Add an equal volume of freshly made 1 M NaOH to the DNA samples. 9. Incubate the DNA solution at room temperature for 20 minutes. 10. Apply the DNA solutions to the dot blot apparatus according to the manufacture's instructions. Allow the DNA to incubate with the membrane at room temperature for 10 minutes. 11. Apply a vacuum to the dot blot apparatus to draw the DNA solution through the membrane. 12. After the DNA solution has been drawn through, remove the membrane from the apparatus. 13. Incubate the membrane in approximately 100 ml of Neutralizing solution for 30 minutes. 14. Rinse the membrane in 2X SSC and air-dry before use in the hybridization step. 15. To hybridize a probe to the membrane, see Protocol ID#569.
Solutions Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Neutralizing Solution    1 mM EDTA 1.5 M NaCl 0.5 M Tris, pH 7.2 1% (w/v) SDS 2X SSC    30 mM Sodium Citrate, pH 7.0 0.3 M NaCl 1 M NaOH    Make fresh before use
BioReagents and Chemicals Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
Sodium Hydroxide SDS Tris EDTA Sodium Citrate Sodium Chloride
Protocol Hints Title | Overview | Procedure | Solutions | BioChemicals | Hints | Printable Version
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