DNA Digests in Agarose Plugs
Decant equilibration buffer and
wash with digestion buffer 2 times for 15 minutes
Decant digestion buffer wash and
add a fresh 200Ál.
Add enzyme (typically 40 U).
Mix gently and incubate at appropriate
temperature for at least 4 hours.
It may be necessary to remove the
enzymes prior to Pulsed Field Gel Electrophoresis
Remove plug from digestion buffer
and incubate in ESP at 50oC for 2 hours.
Wash twice for 15 minutes in PFGE
Load and run PFGE gel.
Cut plugs as required.
Incubate with gentle shaking for
1 hour in a petri plate with dH2O to
remove storage EDTA.
Equilibrate plugs for 30 minutes
in restriction enzyme buffer.
Prepare digestion buffer
400Ál 10X buffer
2Ál of 1M DTT
80Ál of 0.1M spermidine
20Ál 20mg/ml BSA.
ESP: Dissolve 1 gram SDS in 100 ml 0.5M EDTA
(pH 9.0). Add 100mg proteinase K. Store at -20oC
in 5ml aliquots.