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
   • 400µl 10X buffer
   • 2µl of 1M DTT
   • 80µl of 0.1M spermidine
   • 20µl 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 200µl.
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 50^oC 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 -20^oC in 5ml aliquots.