Dig high prime dna labeling and detection starter kit 1

Core particles associated with HBV DNA replicative intermediates were extracted from HepG2.2.15-CD36OE and HepG2.2.15-vector cells after 96 hours, according to the previously described method[13 (link)]. HBV DNA samples were separated by electrophoresis in 1% agarose gel and transferred onto nylon membranes. UV cross-linking, prehybridization, and hybridization were performed according to the protocol from the manufacturer of the DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche, Germany). The signal was detected by exposure on an X-ray film.

All purified plasmids containing clone sequences were quantified in NanoVue PlusTM (GE Healthcare, Princeton, NJ, USA) and then diluted to a final concentration of 50 ng/μL. These plasmids were denatured by heating to 100 °C for 7 min and then quickly chilled in an ice/water for 10 min. The denatured plasmids were transferred onto the Amersham Hybond-NC nylon membrane (GE Healthcare, Life Sciences, Indianapolis, IN, USA). Then, 1 μL of each plasmid was spotted onto the membranes, and DNA was fixed to the membrane by UV crosslink using a StratalinkerTM UV Crosslinker (Stratagene, La Jolla, CA, USA). After fixation, the membrane underwent pre-hybridization for 30 min. The T. arundinaceum gDNA probe was labeled with digoxigenin-11-dUTP (DIG) using a DIG Nick Translation Kit (Roche Diagnostics). Hybridization was performed as described in the Instruction Manual of the DIG High Prime DNA Labeling and Detection Starter Kit I (Roche Diagnostics). Hybridization signals were subsequently detected and quantified by using the AxioVision measurement module of the Carl Zeiss Scope.A1 Imager fluorescent microscope (Carl Zeiss, Gottingen, Germany). Adobe Photoshop 6.0 was used to adjust the pictures.

PCR-based replicon typing (PBRT) was performed on transconjugants and transformants using primers as described previously (Carattoli et al., 2005 (link)). Then PFGE with S1 nuclease (Takara Biotechnology, Dalian, China) digestion of whole genomic DNA was carried out as described previously (Barton et al., 1995 (link)). The resulting gels were analyzed by Southern transfer and probing with a DIG-labeled bla_CTX-M gene fragment according to the manufacturer’s instructions (DIG High Prime DNA Labeling and Detection Starter Kit I, Roche Applied Science, Mannheim, Germany).

Genomic DNA was isolated from leaves of C. intermedia using CTAB method (Porebski, Bailey & Baum, 1997 (link)), and 20  µg DNAs were digested with BamH I and EcoR V, respectively. The obtaining fragments were separated by electrophoresis through a 1.0% agarose, then the DIG-labeled DNA probes of CiGAD1 and CiGAD2 were hybridized followed the protocol of DIG-High Prime DNA Labeling and Detection Starter Kit I (Roche Applied Science, Mannheim, Germany). Briefly, the DIG-labeled DNA probe was prehybridized at 60 °C for 2 h and then hybridized 37 °C for 12 h. Following hybridization, the blot was washed twice in 2 × SSC containing 0.1% SDS at 25 °C for 5 min and was washed twice in 0.1 × SSC containing 0.1% SDS at 50 °C for 15 min. The immunodetection of the DIG-labeled probe was performed with 1:10,000 anti DIG-AP. The blot was exposed to X-ray film (AGFA, Germany). DNA probes were amplified from genomic DNA by using the primers designed in the sequenced DNA fragments based on the cDNA sequences of CiGAD1 and CiGAD2, while the probe for CiGAD1 was mainly in the region from 1,076–1,370 bp in the full-text cDNA, and the probe for CiGAD2 was mainly in the region from 639–1,031 bp in the full-text cDNA. Primers are listed in Table S1 and probe sequences are listed in Supplemental Information 3.

Conjugation experiments of bla_CTX-M-55 gene positive Salmonella isolates were conducted by liquid mating in LB broth using sodium azide-resistant E. coli J53 as the recipient strain. Transconjugants were selected on MacConkey agar containing cefotaxime (2 ug/mL) and sodium azide (300 ug/mL). The presence of bla_CTX-M-55 in transconjugants was verified by PCR and sequencing as described. PFGE analysis was conducted using S1 nuclease (Takara Biotechnology, Dalian, China) digested genomic DNA as previously described (Barton et al., 1995 (link)) to identify the genetic location of bla_CTX-M genes. Primers used for bla_CTX-M-probes were the same as those used to amplify CTX-M encoding genes. The resulting gels were analyzed by Southern blotting after transfer to Hybond-N+ membranes (GE Healthcare, Little Chalfont, United Kingdom) and probing with a DIG-labeled bla_CTX-M gene fragment according to the manufacturer’s instructions (DIG High Prime DNA Labeling and Detection Starter Kit I, Roche Applied Science, Mannheim, Germany). Restriction fragments of agarose-embedded DNA of strain H9812 digested with XbaI(Takara) at 37°C for 4 h was used as DNA size marker during electrophoresis.

To construct the MaAreB-disruption vectors, about 1000-bp up- and down-stream fragments of MaAreB were cloned from WT genomic DNA and inserted into pK2-SM-F and pK2-SM-R vectors [30 (link)] to form the gene knockout vectors, pK2-SM-MaAreB-F and pK2-SM-MaAreB-R (Figure S1A). The DNA and promoter sequence of MaAreB was amplified and inserted into pK2-sur vector [31 (link)] to form the complementation plasmid, pK2-sur-MaAreB (Figure S1B). Both knockout and complementation recombinant vectors were transferred into AGL1 for fungal transformation [32 (link)]. The ΔMaAreB and CP transformants were screened on CZA plates supplemented with 0.5 g/L glufosinate-ammonium (Sigma, St. Louis, MO, USA) or 0.02 g/L chlorimuron ethyl (Sigma, Bellefonte, PA, USA), respectively. Firstly, the fragments of target gene and resistance genes were amplified for preliminary verification of the transformants. On the other hand, Southern blotting was used for further verification via DIG High Prime DNA Labeling and Detection Starter Kit I (Roche, Basel, Switzerland) (Figure S1C). All primers are listed in Table S1.