Dig high primer dna labeling and detection starter kit 1

According to the method described by Ding et al. [21 (link)], Ab-far-n hpRNA transgenic B. cinerea were obtained and cultured at 25 °C for later use in subsequent experiments. Genomic DNA of transgenic B. cinerea was extracted from the hygromycin-tolerant generation, as described previously [40 (link)]. Ab-far-n (n: 1–8) hpRNA transgenic B. cinerea (ARTBn) were selected and identified by PCR with the primers (Table S2) and used as the control. The PCR fragments obtained were sequenced to ensure accuracy. For Southern blot analysis, the primers, Hph-DIG-F/Hph-DIG-R (Table 1), were designed to amplify the DIG-labeled probes. Approximately 10 μg genomic DNA from ARTBn was digested with EcoRI. The digested DNA was separated on a 0.8% agarose gel and then transferred to a Hybond-N+ membrane (Amersham, Little Chalfont, UK) [41 (link),42 (link)]. Hybridization and detection were performed with a Dig High Primer DNA Labeling and Detection Starter Kit I (Roche, Basel, Switzerland) according to the manufacturer’s instructions. Equal amounts of genomic DNA from WTB were used as controls.

A total of 10 μg genomic DNA was denatured by heating and blotted onto a Hybond TM-XL membrane (Amersham, Bath, UK) soaked with 2× SSC. The membrane was then dried and baked at 80°C for 2 h. Hybridization was performed according to the manufacturer’s protocol for the Dig High Primer DNA Labeling and Detection Starter Kit I (Roche, Basel, Switzerland)

Primers in Table 1 were designed to amplify the Digoxigenin (DIG)-labeled probe with a PCR DIG Probe Synthesis Kit (Roche, Basel, Switzerland). Approximately 20 μg of gDNA was obtained from CFN and digested with restriction enzymes (Dra I and EcoR I) (Figure S4). The digested DNA products were separated by 0.8% (w/v) agarose gel electrophoresis and transferred to a Hybond N⁺ membrane (Amersham-Biosciences, Little Chalfont Buckinghamshire, England) [27 (link),40 (link)]. The membrane was hybridized for 18 h at 42 °C with the probe. Hybridization was performed using a Dig High Primer DNA Labeling and Detection Starter Kit I (Roche, Basel, Switzerland) according to the manufacturer’s instructions. After hybridization, the membrane was washed with 2 × SSC/0.1% SDS for 15 min at 25 °C followed by 0.5 × SSC/0.1% SDS for 30 min at 65 °C and examined. Equal amounts of carrot callus gDNA and plasmid pET-32a-Ar-far-1 were used as a control group [39 (link)].

The Southern-F and Southern-R primers (Table 1) were designed to amplify a 378-bp digoxigenin (DIG)-labelled probe using the PCR DIG Probe Synthesis Kit (Roche, Germany). Approximately 10 μg of R. similis gDNA was digested with EcoR I and Xba I. The digested DNA was separated via electrophoresis and then transferred to a Hybond N⁺ membrane (Amersham) [38 (link)]. Hybridization and detection were carried out using the Dig High Primer DNA Labeling and Detection Starter Kit I (Roche). An equal amount of gDNA from carrot callus was used as a control.

Genomic DNA was isolated from the tender leaves of T₀, T₁ and T₂ rice seedlings using the Cetyltrimethylammonium bromide (CTAB) method [18 ]. The primer pairs P1/P7 and P3/P8 were designed to amplify mohrip1 and mohrip2, respectively, from transgenic rice (Table 1). Genomic DNA was digested using BamHΙ and EcoRΙ, separated via 1.2% agarose gel electrophoresis, and then transferred onto Hybond-N nylon membranes (Amersham, Uppsala, Sweden) for Southern blotting [19 (link)]. The MoHrip1 and MoHrip2 probes used in the experiment were labeled using the Dig-High primer method. Hybridization was performed according to the Dig-High Primer DNA Labeling and Detection Starter Kit I (Roche, Germany).

The primers 179SB-F/179SB-R (Table 1) were designed to amplify a DIG-labelled probe using a PCR DIG Probe Synthesis Kit (Roche Applied Science, Penzberg, Germany). Approximately 10 μg of R. similis gDNA was digested with EcoR I and Hind III (Thermo Fisher) overnight at 37 °C. The digested DNA was separated via electrophoresis and transferred to a Hybond N⁺ membrane (Amersham Biosciences, GE Healthcare, UK). Hybridization and detection were performed using a Dig High Primer DNA Labeling and Detection Starter Kit I (Roche Applied Science) at 48 °C for 24 h. pTA2-gscp digested with EcoR I and Hind III was used as a control.

To determine the copy number of the Tn5 transposon in mutant strains, total DNA was digested with EcoRI, PstI, and BamHI separated by electrophoresis on 0.8% (w/v) agarose gel and transferred onto nylon membranes (Hybond-N+; Amersham, GE Healthcare, Piscataway, NJ, United States). A digoxigenin-labeled, kanamycin-resistant, gene probe (Xia et al., 2015 (link)) was used to do hybridization and detection according to the protocol for the DIG High Primer DNA Labeling and Detection Starter Kit I (Roche).
Shotgun cloning was performed to determine the transposon insertion site. Chromosomal DNA samples were restricted with PstI and EcoRI and ligated into pBluescript II SK. E. coli DH5α transformants were selected on LB medium that contained kanamycin. Positive clones were sequenced with Tn5-39 and Tn5-1571 (Xia et al., 2015 (link)) to allow determination of the precise location of a transposon insertion. Sequences were then compared to the protein sequence database (GenBank) using the BlastX algorithm (Altschul et al., 1990 (link)). For each mutant, the joins between the transposon sequences were identified.