Certified low melt agarose

The successful linearization of the BACs has been assessed through Pulse Field Gel Electrophoresis (PFGE) using the Clamped Homogeneous Electric Fields (CHEF) technology. 2% low melting agarose solution (Certified TM Low Melt Agarose, BioRad) was prepared using 1X TBE buffer (Tris base 1M, Boric Acid 1M, EDTA 0,02M) and melted using a microwave. The solution was equilibrated at 50°C in a water bath. 1 µg of linearized BAC DNA was combined with equal volume of 2% Certified TM Low Melt Agarose and mixed by pipetting in order to achieve a final concentration of 1% agarose. The mixture was quickly transferred to plug molds and left to solidify for 10 minutes. While waiting for the solidification of the plugs, 1% agarose gel (Pulse Field Certified Agarose, BioRad) was prepared using 1X TBE buffer and casted in a PFGE casting stand. Finally, the plugs were inserted inside the wells. PFGE was run according to the following program for 20 hours: The analysis was conducted with two separate biological replicates. The conditions used for the qPCR were 95°C 1 min initial denaturation, 95°C 30 s, 65°C 30 s, 72°C 30 s for 40 cycles with fluorescence detection after every elongation step. The PCR products were not longer than 250 bp and contained a GC content of ∼50%. The experiment was performed on an Eppendorf Realplex 2 Mastercycler.