Extract n amp kit

To determine embryo genotypes, embryos were collected after imaging and genomic DNA extracted using the Extract-N-Amp kit (Sigma) in a final volume of 10 µl. Genomic extracts (1–2 µl) were then subjected to PCR using allele-specific primers (Supplementary file 3).

The embryos generated from pairwise breeding of single gene and multi-gene heterozygote mutant fish were grown to adulthood (3–6 months). Fin clips from adult fish were processed for DNA extraction using the “Extract-N-Amp” kit (Sigma-Aldrich) and used for genotyping by fluorescent PCR method as described [67 (link)]. The genotyping data were used to analyze for Mendelian ratios of surviving homozygous knockout fish compared to the homozygote WT and heterozygous fish. Under the null hypothesis of no viability selection, progeny genotypes should conform to an expected Mendelian ratio of 1:2:1. Deviations from expected number of homozygous knockouts (25%) were tested with goodness-of-fit Chi-square statistical analysis. If the parent fish were heterozygote for mutations in more than one gene, data were analyzed for survival of all possible genotypes expected from these breeding. To get sufficient number of fish genotyped, we analyzed progenies from two breeding for most alleles. To determine the presence of both sexes among surviving adults, all genotyped fish were categorized as males and females and counted.

The Amelx promoter-Mmp20 cDNA with the 3′-Amelx non-coding region was excised from the vector by restriction digestion with NotI-SrfI, purified with a Qiaquick gel extraction kit (Qiagen, Germantown, MD, USA) and microinjected into fertilized oocytes for surgical transfer to recipients. Germline transmission was determined by PCR analyses of genomic DNA obtained from tail biopsies. PCR primers used to identify the presence of the transgene were: 5′-GAA AAT GGT TTG CAG CAT CA-3′, and 5′-CTT GCC ACC ATC TCG CCA GCC-3′. For mouse genotyping, genomic DNA was isolated from tails and the Extract-N-Amp Kit (Sigma) was used for PCR reactions. PCR primer sequences for determining Mmp20 genotypes were: 5′-CTG CGT CCC CAG ACT TTT GAT TT-3′, and 5′-GCT TTT CAT GGC CAG AAT GCT CT-3′, to detect the ablated allele and 5′-AAG TAG ACT GAA GTC AGG AGA GCC-3′, and 5′-CTG TAG TGG TGA CCC TAG TCA TCT T-3′, to detect the wild-type allele. Offspring carrying the Mmp20 transgene (Tg) were mated with Mmp20 ablated mice [22] (link) to produce Tg positive Mmp20^+/− and these mice were bred to generate both Tg positive Mmp20^−/− and Tg positive Mmp20^+/+ mice. Therefore, each transgenic founder mouse line had a similar genetic background.

Isolates with dark black growth were considered viable and allowed to grow at 20°C until ~5 mm diameter (approx. 6 weeks to 3 months). Colonies were transferred onto a cellulose grid filter (GN Metricel 28148–813) on MMN plates using the previous protocol except adding 7 g l^-1 dextrose and omitting antibiotics and allowed to grow for 1–3 months for DNA extraction. The Extract-N-Amp kit (Sigma-Aldrich XNAP2-1KT) was used to extract genomic DNA, following manufacturer instructions except the modification to use only 20 μL of the Extraction and Dilution solutions [45 (link)]. DNA samples were stored at -20°C until use in PCR and sequencing efforts below.

RyR1 I4895T knock-in mice on a 129S2/SvPasCrl background were kindly provided by the S. Hamilton laboratory, Texas, USA. These founder mice were rederived using C57BL/6J for breeding. This resulted in a new sub-line with a mixed genetic background (approximately 50% 129S2/SvPasCrl and 50% C57BL/6J after rederivation). Since homozygotes have a perinatal lethal phenotype (44 (link)), heterozygotes (HET) were crossed with wild-type (WT) littermates or C57BL/6J mice (Charles River) to maintain the colony. At least five such crosses had occurred before the experiments. Ear snips from adult mice and tail snips from sacrificed neonates were retained for genotyping. DNA was extracted from tissue samples using the Extract-N-Amp kit (Sigma) and PCR was used to amplify the fragment covering the mutation site. Forward and reverse primers were 5′-GGTCTTCCTGTCTCAATAACCCGATCTAGAAAC-3′ and 5′-GATGGAGAAACCAAAGCTCAGAGAGACCAC-3′, respectively. Since the inserted mutation site also contains an Age I target sequence, only samples containing the mutated allele undergo Age I digestion.