Gel doctm ez imager

Genomic DNA was isolated from wild-type and putative transgenic rice calli using the ZR Genomic DNA Kit (Zymo Research Corp, Irvine, CA, USA). The quality of genomic DNA was checked on 1.0% agarose gel before doing genomic DNA PCR. The presence of rhAA was determined using gene-specific forward and reverse primers, which were used for the construction of the recombinant plant expression vectors. PCR was carried out in a 20 μL volume containing 100 ng of genomic DNA, 10 pmol of the primer sets specific for the target genes, and 10 μL of 2× GoTaq master mix containing dNTPs and polymerase. PCR was processed as follows: 95 °C for 5 min, followed by 30 cycles of (95 °C for 1 min, 58 °C for 1 min 30 s, and 72 °C for 1 min), and final extension at 72 °C for 2 min. PCR products were analyzed by 1.0% agarose gel electrophoresis and visualized by ethidium bromide staining under ultraviolet light using a Gel Doc^TM EZ Imager (Bio-Rad Laboratories, Inc., Hercules, CA, USA).

The Wizard^TM DNA Purification Kit (Promega) was used to extract the genomic DNA from the fresh, pure colonies of S. aureus as per the manufacturer's specifications. The universal primers 27 F 5′-AGAGTTTGATCCTGGCTCAG-3′ and 1492 R 5′-GGTTACCTTGTTACGACTT-3′ were used for DNA amplification. The amplified DNA fragments were examined by agarose gel electrophoresis and observed with the Gel Doc^TM EZ Imager (Biorad). The 16S rDNA gene sequences were manually checked and analyzed using the software BioEdit, Version 7.2.6.1 (Tom Hall, Ibis Biosciences, Carlsbad, California, USA). The sequences obtained after analysis were compared to the NCBI (National Center for Biotechnology Information) online database using BLAST (Basic Local Alignment Search Tool) to retrieve similar sequences.

Livestock associated Methicillin resistant S. aureus harboring mecC gene were identified by conducting PCR on all phenotypically identified MRSA isolates with positive growth on MRSA selective agar, ORSAB. Specific primers for mecC genes (Table 1) as described earlier [21 (link)] were used to identify mecC positive LA-MRSA isolates. Two microliters of sample were added to 48 µL of master mix containing 26.5 µL nuclease free water, 10 µL 5X buffer, 2 µL 50 mM MgCl2, 1 µL 10 mM dNTPs, 3.75 µL for both 10 µM mecC R and mecC F and 1 µL Taq DNA polymerase (5 u/µL). The PCR protocol was set as pre-denaturation at 95 °C for 2 min, 30 cycles of amplification with denaturation at 95 °C for 45 s, annealing at 55 °C for 1 min, extension at 72 °C for 2 min and final extension at 72 °C for 5 min. The PCR products were analyzed by gel electrophoresis using 1.2% agarose and gel imaging was done using Gel Doc^TM EZ Imager (Bio-Rad, Hercules, CA, USA). The expected amplification product of 304 bp signifies a positive detection of mecC gene.

In order to purify the LAMP products from positive reactions, a Gel / PCR Purification kit (Yekta Tajhiz, Tehran, Iran) was used according to the manufacturer’s instructions. Purified amplicons were digested with 8 units of the restriction endonuclease AluI (New England BioLabs, Ipswich, MA, United States) at 37 °C for 60 min. Purified amplicons and digested products were electrophoresed on 3% gel agarose, stained with FluoroDye DNA Fluorescent Loading Dye 10000X (SMOBIO, Hsinchu, Taiwan) and finally visualized using a GelDoc ^TM EZ Imager (Bio-Rad Laboratories, Inc., California, United States). In addition, the LAMP products were also sequenced to validate the accuracy of the LAMP assay.

Oocyte mRNA was isolated using a Dynabeads mRNA DIRECT kit (Dynal Asa, Oslo, Norway) according to the manufacturer’s instructions. Briefly, oocytes were suspended with lysis/binding buffer and mixed with prewashed Dynabeads oligo dT₂₅. After mRNA binding, the beads were washed with buffer A twice, followed by buffer B, and mRNA was eluted with Tris-HCl by incubation at 73 °C. Purified mRNA was used as a template for reverse transcription with an oligo (dT) primer according to the MMLV protocol. RT-PCR was performed with single oocyte-equivalent cDNA and primers (Table 2). Then, the RT-PCR products were separated by electrophoresis on a 1.5% agarose gel and analyzed using a Gel Doc^TM EZ Imager (Bio-Rad, Hercules, CA, USA). All experiments were repeated three times.

Except for the ITS region of the rRNA gene, all the 305 E. bieneusi DNA specimens were also analyzed at the four MLST loci, including three microsatellites (MS1, MS3, and MS7) and one minisatellite (MS4). The primers and the cycle parameters for nested PCR amplifications used in the present study were designed previously by Feng et al. (2011) (link) and the approximately expected fragment lengths of the secondary PCR products were 676 for MS1, 537 for MS3, 885 for MS4 and 471 for MS7 (Feng et al., 2011 (link)). TaKaRa Taq DNA polymerase (TaKaRa Bio Inc., Tokyo, Japan) was used in all the PCR reactions. All secondary PCR products were separated by electrophoresis in a 1.5% agarose gel and visualized on GelDoc^TM EZ Imager (Bio-Rad, United States) by staining the gel with GelStrain (TransGen Biotech., Beijing, China) before sequencing.