Redextract n amp kit

Genetic profiles were generated by PCR using the primer M13 (5′-GAGGGTGGCGGTTCT-3′)⁸⁵ . DNA was extracted from all of the isolates using a REDExtract-N.Amp kit (Sigma) and amplified following the manufacturer’s recommendations to give a final volume of 20 μl per reaction; the thermal cycling parameters were: 7 min at 95 °C, 35 cycles of 1 min at 94 °C, 1 min at 45 °C and 2 min at 72 °C, followed by a 6 min final extension at 72 °C. A 1.5% agarose gel containing ethidium bromide was loaded with 5 µl of each of the PCR products and electrophoresis run at 85 V for 90 minutes in freshly prepared 1x TBE-EDTA buffer at pH 8.0 using a Bio-Rad PowerPac 300 power supply; a DNA molecular weight marker (1 kbp) was used as a molecular size standard. Photographs of the electrophoresis results recovered as TIFF files were aligned using BioNumerics package 6.0 into similarity groups.

Total genomic DNA was extracted with the REDExtract-N-Amp Kit (Sigma-Aldrich) following the manufacturer's instructions. The mitochondrial CR and the first intron of the nuclear S7 ribosomal protein gene (S7) were amplified using L-pro1 and H-DL1 [35 (link)], and S7RPEX1F and S7RPEX2R [36 (link)], respectively (see electronic supplementary material, appendix I for details).
Sequences were edited with Codon Code Aligner (Codon Code Corporation) and aligned with Clustal X 2.1 [37 (link)]. Whenever possible, both strands of the same specimen were recovered for S7 following the approach of [38 (link)]. Sequences obtained were deposited in GenBank (accession nos. KU751889-KU752182). Additional sequences available in GenBank were obtained from [32 ,33 ] (electronic supplementary material, table S1 provides the original reference for each individual sequence).

Mouse genomic DNA was extracted from tail biopsies by using a tissue PCR Kit (REDExtract-N-Amp kit; Sigma-Aldrich), according to the manufacturer’s protocol. Two female founders were identified by Sanger sequencing of a PCR product flanking the targeted mutation site generated with the following primer pair: Tgm2—right: 5′-GAC TTT GAT CCC TTG CCG TA-3′ and Tgm2—left: 5′-GTC CAC CCA GAG ACT GGA AA-3′. For subsequent generations, an ARMS PCR assay was performed as follows: the Tgm2 WT allele was identified using Tgm2—right 5′-GAC TTT GAT CCC TTG CCG TA-3′ and Tgm2 WT—left 5′-GCT GCA AAC ACC CAG CA-3′. The mutant genotype was identified using Tgm2—right: 5′-GAC TTT GAT CCC TTG CCG TA-3′ and Tgm2-C277S mutant—left: 5′-GCT GCA AAC ACC CAG CT-3′. PCR conditions were as follows: initial denaturation at 94 °C for 30 s, 40 cycles at 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 40 s, followed by final extension at 72 °C for 30 s. Both mutant and WT PCR protocols were performed for each tail biopsy. The presence of WT amplicon alone indicated a WT mouse, the presence of the mutant band alone indicated a homozygous mutant, and the presence of both indicated a heterozygous mouse.