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HOME > Protocols > Molecular Biology > DNA > Protocol for Genomic DNA Digests in Agarose Plugs

Genomic DNA Digests in Agarose Plugs

  1. Cut plugs as required.
  2. Incubate with gentle shaking for 1 hour in a petri plate with dH2O to remove storage EDTA.
  3. Equilibrate plugs for 30 minutes in restriction enzyme buffer.
  4. Prepare digestion buffer
      • 3.5ml H2O
      • 400Ál 10X buffer
      • 2Ál of 1M DTT
      • 80Ál of 0.1M spermidine
      • 20Ál 20mg/ml BSA.
  5. Decant equilibration buffer and wash with digestion buffer 2 times for 15 minutes each.
  6. Decant digestion buffer wash and add a fresh 200Ál.
  7. Add enzyme (typically 40 U).
  8. Mix gently and incubate at appropriate temperature for at least 4 hours.
  9. It may be necessary to remove the enzymes prior to Pulsed Field Gel Electrophoresis (PFGE).
  10. Remove plug from digestion buffer and incubate in ESP at 50oC for 2 hours.
  11. Wash twice for 15 minutes in PFGE buffer.
  12. Load and run PFGE gel.

ESP: Dissolve 1 gram SDS in 100 ml 0.5M EDTA (pH 9.0). Add 100mg proteinase K. Store at -20oC in 5ml aliquots.

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