Bgl 2 endonuclease

Adult F₁ fish with mutations detected by PCR were sequenced to confirm DNA sequence changes of the targeted genes. The gDNA sequences around the targeted regions were amplified using primers for T7E1 assays by high fidelity FlashPfu DNA Polymerase (Tonk Bioscience). PCR products were purified with NucleoSpin Gel and PCR Clean-Up (Takara Bio) according to its manufactory manual. The purified PCR products were ligated with the linearized pJet1.2 vector by T4 DNA ligase from CloneJET PCR Cloning Kit (Thermo Scientific). The ligation reaction was transformed into Top10 E. coli by heat shock. Single isolated colonies were chosen for plasmid mini-preparation and Bgl II endonuclease (New England Biolabs Inc.) diagnosis since the vector has two Bgl II cutting sites at both ends. Three positive plasmid clones were sequenced at the Purdue Genomics Core Facility by T7 sequencing primer.

There were four oligonucleotide combinations used: a) In order to initially validate each primer pair, each of the short and long pairs (21.1–21.10) were tested independently in simplex at a concentration of 250 nM each. Additionally, pair 6.1 was included in each reaction to normalize DNA loading concentration. b) Short pairs 21.1 to 21.10 were sequentially added to pair 6.1 at a final concentration of 250 nM each for short multiplexing experiments. c) For long multiplexing experiments, long pairs 21.1 to 21.10 were sequentially added to pair 6.1 at a final concentration of 50 nM each. The two universal primers were also added at a final concentration of 1 µM each. d) The repetitive sequence pair was added to short pair 6.1, all at a concentration of 1 µM each. Additionally, genomic DNA for this combination was pre-treated with BglII endonuclease (New England BioLabs) in the recommended buffer for 10 min at 37°C.