Pcr cleanup kit

Genomic DNA of the wsp3 and wsp5 strains was isolated using a Zymogen DNA isolation kit (Cat No. D6105, ZYMO RESEARCH), and the 16S rRNA gene sequences were amplified by PCR. The amplified products were run on 1.0% agarose gels and further extracted using a PCR clean‐up kit (Thermo Scientific, Cat No. K0701). The taxonomic identification and percentage similarity of the amplified 16S rRNA gene sequences were calculated using the ‘Identity’ option of the EzTaxon e‐server (http://www.eztaxon.org). The evolutionary history was inferred using the neighbour‐joining method (Saitou and Nei, 1987). The optimal tree with the sum of branch length = 1.21597215 is shown. The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1500 replicates) are shown next to the branches (Felsenstein, 1985). The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Poisson correction method and are in the units of the number of amino acid substitutions per site. The analysis involved 18 amino acid sequences. All positions containing gaps and missing data were eliminated. There were a total of 126 positions in the final data set. Evolutionary analyses were conducted in MEGA7 (Kumar et al., 2016).

The amino acid sites selected for mutation are shown in Table 3. KOD FX DNA polymerase (Toyobo) was applied to construct the mutated genes from the wild-type pET28-flaB template via thermal cycling at 95°C for 3 min followed by 25 cycles at 95°C for 15 s, 62°C for 15 s, 68°C for 1 min, and a final extension at 72°C for 5 min. FastDigest DpnI (Thermo Fisher) was added to the PCR product and incubated in a 37°C water bath for 2 h. The mixture was purified using a PCR Clean-Up Kit and transformed into DMT competent cells. Transformants were selected for sequencing using T7 promoter and T7 terminator primers.

The guide RNA sequence (5′-GGCTTTGCATTGCGAGCTGT-3′) was designed to target DNA sequence downstream of the Pramef12 start codon. Double-stranded synthetic DNA was cloned into pDR274 (Addgene, #42250) expressing a single guide RNA (sgRNA). After linearization by digestion with DraI, the DNA fragment was purified with PCR Clean-up Kit (Clontech Laboratories) and transcribed using AmpliScribe T7-Flash Transcription Kit (Lucigen). Cas9 mRNA (Addgene #42251) was generated by linearization with PmeI, purified with PCR Clean-up Kit, and transcribed with mMESSAGE mMACHINE T7 Kit (Thermo Fisher Scientific). After transcription, the sgRNA and Cas9 mRNA were purified with MEGAclear Transcription Clean-Up Kit (Thermo Fisher Scientific). Hormonally stimulated B6D2_F1 (C57LB/6 × DBA2) female mice were mated to B6D2_F1 male mice and zygotes were collected from oviducts at embryonic day 0.5 (E0.5). sgRNA (50 ng μl⁻¹) and Cas9 mRNA (100 ng μl⁻¹) were mixed and injected into the zygotes in M2 medium. Injected zygotes were cultured in KSOM (37 °C, 5% CO₂) to the two-cell embryo stage. Two-cell embryos were transferred into the oviducts of pseudo-pregnant ICR female mice at E0.5. Primers for genotyping the Pramef12^Null mice are listed in Supplementary Table 1. Uncropped gels are presented in Supplementary Fig. 8.

Colony PCR was performed using the primers 341F (5′-CCTACGGGNGGCWGCAG-3′) and 805R (5′-GACTACHVGGGTATCTAATCC-3′) for the V3–V4 region using the GoTaq Green Mix containing the GreenTaq polymerase from Genescript Inc. PCR conditions included denaturation at 95 °C for 5 min, followed by 30 cycles at 94 °C for 1 min, 50 °C for 1 min (annealing), and 72 °C for 1 min, with a final extension at 72 °C for 10 min. The amplified products were visualized by electrophoresis on a 1% agarose gel pre-stained with ethidium bromide, run at 70 V for 45 min, and using 1× TAE as the running buffer in a model 250 electrophoresis chamber (Life Technologies, Carlsbad, CA, USA). The 1 kb Plus DNA ladder (Invitrogen, Waltham, MA, USA) was added as a standard. The amplified products were purified using a PCR cleanup kit (Bio Basic Canada Inc., Markham, ON, Canada), quantified with a Nanodrop One microvolume UV-visible spectrophotometer (Thermo Scientific, Waltham, MA, USA), and Sanger sequencing with 341F as the sequencing primer at the Genewiz sequencing facility, South Plainfield, NJ, USA. The generated sequences were compared using the Basic Local Alignment Search Tool (blastn) [52 (link)] to identify the genera using the default parameters. Further taxonomic analysis based on the full 16S sequence and whole genome analysis was performed as described in Section 2.4.