Chef dr 2 drive module

To ensure the isolation of DNA of the expected size, two partial restriction digests were performed to determine the conditions yielding an appropriate percentage of fragments between 100 and 350 kb in size. Size selection was performed according to previously described protocols using the HindIII enzyme (New England Biolabs, UK) [19 (link), 21 ]. The optimal conditions were determined by digesting the chopped plugs at several enzyme concentrations (0.2–50 U). The macerated plug pieces were placed in 1.5-mL Eppendorf tubes containing the appropriate buffer and the HindIII enzyme and incubated on ice for 1 hour. Digestion was performed at 37°C for 10 min, and the reaction was stopped by adding 30 μL of 0.5 M EDTA (pH 8.0).
Partially digested HMW DNA was separated via PFGE (pulsed-field gel electrophoresis) in 1% TBE agarose (CHEF-DR II drive module, Bio-Rad) using the following parameters: 120° angle, 12°C, 6.0 V/cm, initial switch time = 1.0 sec, final switch time = 40.0 sec, ramping = linear, and running time = 18 hours. Once the optimal HindIII concentration was determined, mass digestion was performed using 13 plugs. The digested HMW DNA was separated into two selected sizes via PFGE, as described by Peterson et al. (2000) [21 ].
The DNA inserts embedded in agarose gel pieces were maintained in 50-mL polypropylene centrifuge tubes containing 1× TAE at 4°C before electroelution.