Tiangel midi purification kit

Each injected zygote was collected at the blastocyst stage and the DNA was extracted with embryo lysis buffer (contining1% NP40, 50 mM Tris-HCl, 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS) at 50 °C for 20 min and 90 °C for 5 min in a BIO-RAD PCR machine. Genomic DNA from BR, WR and KO rabbits was isolated using the TIANamp Genomic DNA Kit (TIANGEN, Beijing, China) according to the manufacturer’s instructions. PCR primers used for amplification and mutation detection are listed in Supplementary Table S1. PCR products were gel purified with TIAN gel Midi Purification Kit (TIANGEN, Beijing, China) and cloned into pGM-T (Tiangen, Beijing, China). Ten positive plasmid clones were sequenced and the sequences were analyzed by DNAMAN software.

The T7E1 assay was performed as previously described. Briefly, PCR products were purified with a TIANgelMidi purification Kit (TIANGEN, Beijing, China) and were denatured and annealed in NEBuffer 2 (NEB) in a thermo cycler. Hybridized PCR products were digested with T7E1 (NEB, M0302L) for 30 minutes at 37 °Cand subjected to 2% agarose gel electrophoresis.

We extracted genomic DNA using the Tiangen Plant Genomic DNA Kit (Tiangen Biotech Co., Beijing, China). For each taxon, we amplified three chloroplast genes (atpA, matK, rbcL) and one non-coding region (trnL–F) separately with standard polymerase chain reaction (PCR). We amplified the atpA region following the primers and procedure of Schuettpelz and Pryer [23 ]. We amplified the matK region using primers (PolypodF1 and PolypodR1) and PCR protocols introduced by the CBoL Plant Barcoding Working Group (http://www.barcodinglife.org/index.php/Public_Primer_PrimerSearch). We amplified the rbcL region using primers 1F and 1351R, following PCR protocol outlined in Hasebe et al. [36 ] and Murakami et al. [37 ]. We amplified the trnL–F region using the primers and PCR protocol described in Wang et al. [38 ]. We have listed complete primer information in Table 1. We purified our PCR products using the Tiangel Midi Purification Kit (Tiangen). We conducted sequencing reactions using the DYEnamic ETDye Terminator Cycle Sequencing Kit (Amersham Pharmacia Biotech) and sequenced the products on an ABI 3730XL genetic analyzer (Applied Biosystems, Foster City, USA). We aligned all the sequences obtained using CLUSTAL X [39 (link)], and adjusted them manually in BioEdit [40 ].

The colon contents were collected from all mice immediately after sacrifice. The V3–V4 region of the 16sRNA gene was sequenced in the 48 content samples. Microbial DNA was extracted from the colon contents using the QIAamp DNA Stool Mini Kit (Qiagen, Hilden, Germany). The microbial V3-V4 region was amplified by PCR using the primers 5′-ACTCCTACGGGAGGCAGCA and 3′-GGACT ACHVGGGTWTCTAAT. Amplicons were extracted from a 2% agarose gel using the TIANgel MIdi Purification Kit (TIANGEN BIOTECH, Beijing, China) and were subsequently purified. For detailed experimental steps on Illumina MiSeq sequencing, please see our previous article (Zhu et al., 2018 (link)); The general data analysis was performed by a commercial company (Novogene, Beijing, China).

The T7E1 assay was performed as previously described (Guschin et al. 2010 (link)). Briefly, PCR products mentioned above were purified with TIANgel Midi Purification Kit (Tiangen) and then denatured and annealed in NEBuffer 2 (NEB, USA) using a thermocycler. Hybridized PCR products were digested with T7 endonuclease I (NEB) for 30 min at 37° and then analyzed with 2% agarose gel electrophoresis.