Total RNAs were isolated from sweet potato samples using TRIzol reagent (Invitrogen, USA) according to the manufacturer's instructions. RNA quality and purity were assessed by agarose gel electrophoresis. The total RNAs were then reversely transcribed with PrimeScriptTMRT Reagent Kit (TaKaRa, Japan) using Oligo(dT) as primer. Cloning primers were designed according to the assembled contig of IbCAT2 in the sweet potato transcriptome [32 (link), 34 (link)] using Primer Premier 5.0 (PREMIER Biosoft International, CA, USA). Polymerase Chain Reaction (PCR) was performed using KOD-Plus-Neo (TOYOBO, Japan) with IbCAT F1 (5′-GATATCATGGATCCTTATCAGCACCG-3′) and IbCAT R1 (5′-GGAATTCTCACATTGTTGGCCGCAC-3′; bp position: 1049–1069) as primers with 35 cycles of 2 min at 94°C, 10 s at 98°C, 30 s at 56°C, and 45 s at 68°C. The PCR product was separated by 1% agarose gel and purified by DNA gel extraction kit (OMEGA, USA). It was then double digested by EcoR I and EcoR V (Fermentas, USA). The pET-32a(+) was also double digested by EcoR I and EcoR V (Fermentas, USA). Restricted DNA products were then separated by 1% agarose gel, purified by DNA gel extraction kit (OMEGA, USA), and then ligated with pET-32a(+) by T4 DNA ligase (TaKaRa, Japan). The recombinant plasmid was transformed into the Escherichia coli host strain JM109. Positive clones were sequenced with an ABI 3730 instrument.
Ecorv
Manufactured by Thermo Fisher Scientific
Sourced in United States, Lithuania
EcoRV is a type II restriction endonuclease enzyme that recognizes and cleaves the palindromic DNA sequence 5'-GATATC-3'. It is commonly used in molecular biology applications such as DNA digestion, fragment analysis, and genetic engineering.
Automatically generated - may contain errors
Lab products found in correlation
Ecori, by Thermo Fisher Scientific (7 mentions)
Bamhi, by Thermo Fisher Scientific (5 mentions)
Ecorii, by Thermo Fisher Scientific (3 mentions)
Csp6i, by Thermo Fisher Scientific (3 mentions)
Bsu15i, by Thermo Fisher Scientific (3 mentions)
Hindiii, by Thermo Fisher Scientific (2 mentions)
Ndei restriction endonucleases, by Thermo Fisher Scientific (2 mentions)
T4 dna ligase, by Takara Bio (1 mentions)
Primer premier 5, by Premier Biosoft (1 mentions)
Kod plus neo, by Toyobo (1 mentions)
Trizol reagent, by Thermo Fisher Scientific (1 mentions)
Primescript rt reagent kit, by Takara Bio (1 mentions)
Geneart geneoptimizer, by Thermo Fisher Scientific (1 mentions)
Mbo 1, by Thermo Fisher Scientific (1 mentions)
Snabi, by Thermo Fisher Scientific (1 mentions)
Pvu 1, by Thermo Fisher Scientific (1 mentions)
Amersham hybond n membrane, by GE Healthcare (1 mentions)
Dig dna labeling kit, by Roche (1 mentions)
Cdp star, by Roche (1 mentions)
Hybond, by Cytiva (1 mentions)
26 protocols using ecorv
1
Cloning and Expression of IbCAT2 from Sweet Potato
2
Cloning and Expression of Cas13bt Proteins
3
Phage DNA Extraction and Analysis
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Aliquots of phage suspension (1011–1012 PFU/ml) were subjected to phenol/chloroform extraction and ethanol precipitation as described by Carlson and Miller [38] . Isolated phage DNA was subsequently used in restriction analysis, for PCR or was subjected to genome sequencing. Restriction digestion was performed with BamHI, EcoRI, EcoRII, EcoRV, HindIII, KpnI, MboI, NheI, NotI, PstI, PvuI, SnaBI, SspI, VspI and XbaI restriction endonucleases (Fermentas) according to the supplier's recommendations. DNA fragments were separated by electrophoresis in a 0.8% agarose gel containing ethidium bromide. Restriction analysis was performed in triplicate to confirm the results.
4
Southern Blot Analysis of Transformants
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The selected transformants were analyzed by Southern hybridization using a previously described method [27 (link)], to demonstrate that the transformation cassettes had homologously integrated at the targeted T. reesei QM6aΔtmus53Δpyr4 loci (Additional file 3 ). To perform this analysis, 25 µg of total genomic DNA from the parental and mutant strains were digested with EcoRV (Fermentas) overnight, and then, this digested DNA was transferred onto GE Healthcare Amersham Hybond-N + membranes (GE). The probe was generated from the PCR-amplified fragment using Tr69957_pRS426_5fw and Tr69957_pyrG_5rv (Additional file 2 ) and labeled using a digoxigenin (DIG) DNA labeling kit (Roche, Mannheim, Germany) by following the manufacturer’s instructions. Labeling, hybridization, and immunological detection were performed using a non-radioactive labeling and immunological detection kit with CDP-Star as the chemiluminescent substrate (Roche, Mannheim, Germany), by following the methods that were previously described [28 (link)].
5
Phage DNA Restriction Profiling
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Phage DNA digestion and agarose gel electrophoresis were carried out as previously established52 (link). Essentially, the genomic DNA of 90 phage isolates was cut separately with EcoRI, EcoRV or HindIII restriction endonucleases (Fermentas) overnight at 37 °C as recommended by the manufacturer. Enzymes were selected based on their ability to recognize and cleave within 6 nucleotide sites of low G-C content (33%), typical for lactococcal phages and their host genomes. The digested DNA fragments were separated on a 0.7% (w/v) agarose gel containing ethidium bromide in 1xTAE buffer (Merck). DNA loading dye with 50% formamide was used to load the samples on the agarose gel. Formamide was added to separate the phage cohesive (cos) ends53 (link). Comparative analysis based on DNA restriction patterns allowed the separation of the phages into distinct restriction groups (8 in total). Representatives from each group were taken for sequencing.
6
Identification of Putative Circular RNAs
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The tdMDA amplicon was randomly digested with the restriction enzymes, SacI, HindIII, SspI, BamHI, EcoRV, and EcoRI (10U/μl, Thermo Scientific, Waltham, MA, USA) for 3 hours at 37°C. The HindIII, SacI digested fragments were purified by GeNei PCR purification kit (Bangalore, Karnataka, India) and cloned in pOK12 or pBluescript II KS (+) vector at the corresponding site at 16°C for 12 hours. After the confirmation by restriction digestion, a total of nine clones were sequenced, seven for N. benthamiana and two for O. sativa. The mapped clone sequences such as HindIII 10, HindIII 33, HindIII 38, and SacI 11 for N. benthamiana and HindIII 1 and HindIII 2 for O. sativa were subjected to prediction for their possibility of forming circRNA in PlantcircBase [41 (link)] (http://ibi.zju.edu.cn/plantcircbase/index.php). Predicted putative circRNA sequences were validated by RT-PCR with divergent primers (Table 1 ) [42 (link)].
7
Phage DNA Extraction and Analysis
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The aliquot of high-titer (1011–1012 PFU/mL) phage suspension was subjected to phenol/chloroform extraction and ethanol precipitation, as described by Carlson and Miller [33 ]. The isolated phage DNA was subsequently used for PCR and restriction analysis, or it was subjected to genome sequencing. The restriction digestion was performed with Bsu15I, Csp6I, DraI, EcoRII, EcoRV, HhaI, MboI and NdeI restriction endonucleases (Thermo Fisher Scientific, Vilnius, Lithuania), according to the supplier’s recommendations. The DNA fragments were separated by electrophoresis in a 0.8% agarose gel containing ethidium bromide. A restriction analysis was performed in triplicate to confirm the results.
8
Genomic DNA Restriction Digestion
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Restriction digestions were prepared in 10 μl volume reaction containing ∼500 ng of genomic DNA, 1 μL of restriction enzyme: AanI, BcuI, BstEII, EcoRV, HaeIII or NsiI (ThermoFisher) and 1 μl of a 10× corresponding reaction buffer. Reactions were performed at 37°C for 1 h. Afterwards, DNA fragments were visualized on a 1% agarose gel using GelRed as the DNA dye in a loading buffer.
9
Random PCR and SPIA Amplification for RNA-Seq
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The DNA was amplified using random PCR as described elsewhere [14 (link)]. The PCR tag-sequence was cleaved away using EcoRV (Thermo Fisher, Waltham, MA, USA). The RNA was reverse-transcribed into cDNA and amplified by single-primer isothermal amplification (SPIA) using the Ovation RNA-Seq V2 kit (NuGEN, San Carlos, CA, USA) according to the manufacturer’s instructions. The random PCR products and the SPIA products were then purified using the GeneJET PCR purification kit (Thermo Fisher, Waltham, MA, USA) according to the manufacturer’s instructions. The concentrations of the purified random PCR products ranged from 18 to 25 ng/µL, and those of the purified SPIA products ranged from 36 to 66 ng/µL. All the products were sequenced at SciLifeLab/Genome Center (Uppsala, Sweden) using the Ion S5 XL system (Thermo Fisher, Waltham, MA, USA) and two 530 chips. The main type of sequencing errors associated with this platform are insertions and deletions (indels).
10
Analyzing DNA Degradation by Eap
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Herring sperm DNA (10164142), phage lambda DNA (D3779), and pBR322 (N3033L) DNA were purchased from Invitrogen (Karlsruhe, Germany), Sigma-Aldrich, and New England Biolabs (Frankfurt, Germany), respectively. To test the influences of a circular or linear state of DNA, and the nature of the DNA end on degradation by Eap, pBR322 was digested with the restriction enzymes BamHI, EcoRV, and PstI (Thermo Fisher), respectively, following the instructions of the manufacturer. The 1.4-kb DNA fragment was amplified by PCR using primer pair MBH492 (5′-CCTGAACAACCTGATGAGCC-3′)/MBH493 (5′-ACCCTATTTTTTCGCCAAGCC-3′), and chromosomal DNA from S. aureus strain Newman (Duthie, 1952 (link)) as template.
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