Gel pcr dna fragment extraction kit

There were two types of chimeric transcripts detected from the RNA-Seq data in W149 due to the deletion of the stop codon in LOC_Os04g52479 but not in Cypress. In one transcript, the exon 3 of LOC_Os04g52479 joined to the exon 2 of the adjacent locus LOC_Os04g52500. The second transcript involved the joining of exon 4 of LOC_Os04g52479 to exon 1 of LOC_Os04g52500. To validate these chimeric transcripts, two pairs of primers were designed: Exon3-52479F (5′-CACTGGCAACAAGCAGGTTGG-3′), Exon2-52500R (5′-CATGTCGGGGTCCAGAGTGTC-3′), Exon4-52479F (5′-GGAGGCAAGTATGCAAAGTTGGC-3′), and Exon1-52500R (5′-CATCAACGGCTTCAAGAATCGAGC-3′). PCR amplification using cDNA from both W149 and Cypress was done using all combinations of forward and reverse primers following a similar PCR condition and profile described earlier. About 48 µL of each PCR sample was mixed with 2.0 µL 10× loading dye, and these were loaded in 1.5% agarose gel and electrophoresed at 120 V for 60 min. Target amplification band sizes (Cypress: No amplification and W149: 283 bp) were cut from the gel and purified using a Gel/PCR DNA fragment Extraction kit (IBI scientific, Dubuque, IA, USA). Samples were sequenced from both directions via Sanger sequencing at the Genomic Facility of the Louisiana State University.

During the in vitro selection experiment, three PCR reactions were carried out for each round. After the cleavage step, real-time PCR was carried out to quantify the cleaved DNA extracted from the gel. Each 20 μl reaction mixture contained 1 μl purified DNA template, 400 nM primer (P1 and P2) and 10 μl of SsoFast EvaGreen Supermix (Bio-Rad). The thermocycling steps were 95°C for 30 s, 95°C for 5 s followed by 55°C for 5 s. To amplify the library, in PCR1, a 50 μl reaction mixture contained the following: 1 μl DNA template, 200 nM of each of P1 and P2, 200 μM dNTP mixture, 1× Taq buffer and 1.25 units of Taq DNA polymerase. The reaction was carried out for 15–20 cycles (94°C for 5 min; 94°C for 30 s, 55°C for 30 s and 72°C for 30 s). A gel/PCR DNA fragment extraction kit (IBI Scientific) was used to purify the PCR1 product. The purified product was used as the template for PCR2. One-tenth of the purified PCR1 product was further amplified for 12 cycles using P3 and P4 as the primers. A 200 μl PCR reaction mixture contains 4 μl diluted template, 250 nM each of P3 and P4, 200 μM dNTP mixture, 1× Taq buffer and 5 units of Taq DNA polymerase. The final PCR2 product was again purified by 10% dPAGE. The single-stranded FAM-labeled DNA was excised from the gel, ethanol precipitated and used as the library for the subsequent round of selection.

Bacterial isolates were identified by 16S rRNA sequencing. DNA was extracted from pure cultures of each isolate using either a crude extraction (boil in water for 10 min) or the DNeasy Blood and Tissue Kit (Qiagen, Carlsbad, CA). PCR was performed in 25 μL reactions using Platinum Hot-Start Master Mix (Thermo Fisher, Waltham, MA) and 0.2 μM the 27F/1492R universal primers (Frank et al., 2008 (link)). PCR conditions were as follows: initial denaturation at 95°C for 5 min, followed by 30 cycles of 95°C for 30 s, 51°C for 30 s, and 72°C for 2 min, and a final extension at 72°C for 10 min. PCR quality and fragment size were verified by gel electrophoresis (1% agarose gel, 10 V/cm). PCR products were cleaned with the Gel/PCR DNA fragment extraction kit (IBI Scientific, Dubuque, IA) and the DNA concentration was quantified using the Qubit 4 (Invitrogen, Carslbad, CA). Cleaned PCR products were sequenced using both 27F and 1492R primers on an ABI 3730 capillary sequencer (Thermo Fisher) by the Oregon State University Center for Genome Research and Biocomputing (CGRB, Corvallis, OR). Consensus sequences were generated from the forward and reverse sequences for each isolate using SeqTrace (Stucky, 2012 (link)). Taxonomy was assigned using the EZBioCloud Database (Yoon et al., 2017 (link)).

For each sample evaluated, amplification of the V4 hypervariable region of the bacterial/archaeal 16S rRNA gene was performed by using the 515F and 806R primer set according to previously described methods and optimized for the Illumina MiSeq platform [41 (link)]. PCR products were tagged with 280 different 12-bp error-correcting Golay barcodes (http://www.earthmicrobiome.org/) [42 (link)]. PCRs were carried out in triplicate 25-μl reactions using 12–300 ng of template DNA, 1X EconoTaq Plus Green Master Mix (Lucigen, Middleton, WI) and 10 μM of each primer. Thermal cycling consisted of an initial denaturing step of 94°C for 3 min followed by 30 cycles of 94°C for 45 s, 50°C for 1 min and 72°C for 90 s, and a final elongation step of 72°C for 10 min. Replicate amplicons were pooled and visualized on 1.2% agarose gels stained with 0.5 mg/ml ethidium bromide, followed by purification using a Gel PCR DNA Fragment Extraction kit (IBI Scientific, Peosta, IA) and used for downstream analysis.
Amplicon concentration and purity were evaluated using a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE) at wavelengths of 260 and 280 nm.

For amplification of the V4 hypervariable region of the bacterial 16S rRNA gene, primers 515F and 806R were used according to a previously described method (12 (link)) optimized for the Illumina MiSeq platform. The Earth Microbiome Project (13 (link)) was used to select 140 different 12-bp error-correcting Golay barcodes for the 16S rRNA PCR, as previously described (12 (link)). The 5′-barcoded amplicons were generated in triplicate using 12–300 ng DNA template, 1× GoTaq Green Master Mix (Promega, Madison, WI, USA), and 10 μM of each primer. The PCR conditions for the 16S rRNA gene consisted of an initial denaturing step of 94°C for 3 min, followed by 35 cycles of 94°C for 45 s, 50°C for 1 min, and 72°C for 90 s, and a final elongation step of 72°C for 10 min. Replicate amplicons were pooled and purified with a Gel PCR DNA Fragment Extraction kit (IBI Scientific) and visualized by electrophoresis through 1.2% (wt/vol) agarose gels stained with 0.5 mg/ml ethidium bromide before sequencing. Blank controls, in which no DNA was added to the reaction, were performed. Purified amplicon DNA was quantified using the Qubit Flurometer (Life Technologies Corporation, Carlsbad, CA, USA).

Sap inoculum and RNA samples from infected plants were used as templates for cDNA synthesis using the Maxima Reverse Transcription kit according to the manufacturer’s protocol (Thermo Fisher Scientific, Waltham, MA, USA). Two-step RT-PCR was performed using random hexamers for cDNA synthesis, four sets of RRV-specific primers shown in (Table 2), and GoTaq^® Green Master Mix (Promega, Madison, WI, USA). Forty PCR cycles were carried out following the initial denaturation at 94 °C for 5 min: denaturation at 94 °C for 30 s, annealing at 60 °C for 20 s, and elongation at 72 °C for 20 s. The final cycle was followed by an extension at 72 °C for 10 min. The PCR amplicons were examined on 2% TBE agarose gels stained with ethidium bromide, and photographed with a ChemiDoc MP imaging system (Bio-Rad Corp, Hercules, CA, USA). PCR amplicons were purified using the Gel/PCR DNA fragment extraction kit (IBI Scientific, Peosta, IA, USA) and sent to Eton Bioscience (San Diego, CA, USA) for sequencing.