Ten mg of genomic DNA was digested overnight with Sac II and Nde I, separated on a 0.8% agarose gel (Bio-Rad, 161-3102), followed by transfer onto a Amersham Hybond N+ nylon membrane (GE Healthcare) and UV cross-linking (Stratagene, UV Stratalinker 1800).
To characterize the BAC inserts, BAC DNA samples were prepared with a Tomtec Quadra 96 model 320 (Tomtec, Hamden, CT) in a 96-well format, digested with Not I, separated on 1% agarose CHEF (Bio-Rad, Hercules, CA) gels at 5 15 sec linear ramp time, 6 V/cm, 14C in 0.5 × TBE buffer for 16 hours and stained with ethidium bromide.
(ii) In case of β-glucosidase-activity, samples were prepared according to (i) and separated on a SDS-PAGE followed by a 1 min washing step in A. dest, 60 min washing in 1% (v/v) Triton X-100 and another step for 1 min in A. dest.
Fifteen microgram of genomic DNA was digested with Nco I and separated on a 1% agarose gel.
Genomic DNA (10 μg) was digested with EcoR I (Fermentas), separated on a 1% agarose gel and transferred to a nylon membrane (Hybond N+, Amersham, GE Healthcare).
The genotypes of non-synonymous SNP rs3733549 in the unrelated individuals in the world-wide panel of Human Genome Diversity Cell Line Panel [33] were obtained by an independent PCR and cut with the diagnostic restriction enzyme Taq I, and separated on an agarose gel.
Samples in stop solution I were separated on native agarose gels.
Briefly, adequate volumes of hemolysate and purified chicken CA-III were separated on 12.5% PhastGel Homogeneous gels and transferred to Immobilon PVDF transfer membrane (Millipore Corp ,Bedford, Mass, USA) using a commercially available transfer system.
After the nine clones were purified and amplified, the prepared plasmid DNA was digested with EcoR I and Xho I and electrophoretically separated on a 0.8% agarose gel to determine if the plasmid with the inserts were successfully prepared.
DNA was prepared using a Gentra® Puregene® kit (Qiagen, Valencia, CA) One microgram of DNA was digested with Hinf II, Rsa I, Alu I, Hae I, and Msp; then separated on 0.5% agarose gels by pulse-field electrophoresis and transferred to positively-charged nylon membrane by vacuum in alkaline buffer.
The PCR products were purified with QIAquick PCR Purification Kit (Qiagen, Germantown, MD), digested with Sac I and then separated on a 1% agarose gel.
