Plasmids encoding precursor RNAs were derived from pTZ∆P5 [27 (link)], which encodes ∆P5 mutant of the Tetrahymena group I intron ribozyme. These plasmids were constructed by PCR-based mutagenesis. The sequences of the constructed plasmids were confirmed using a 4300 DNA analyzer (Li-COR, Lincoln, NE, USA). Precursor RNAs and P5abc RNA were prepared via in vitro transcription with T7 RNA polymerase and PCR-amplified DNA templates. The T7 promoter sequence was attached via PCR with the T7 promoter-containing sense primer. In vitro transcription reactions of ∆P5 ribozyme precursors were performed in the presence of 5 mM Mg2+ ions and 4 mM rNTPs. Transcription of P5abc RNA was performed with 15 mM Mg2+ ions and 4 mM rNTPs. Each transcription reaction was performed for 4.5 h at 37 °C. The DNA template in the reaction mixture was degraded using DNase I. Crude transcript was purified on a 4% or 6% denaturing polyacrylamide gel. 3′-End labeling of purified RNAs with the BODIPY fluorophore was carried out according to a previously reported procedure [28 (link)].
4300 dna analyzer
Manufactured by LI COR
Sourced in United States, Germany
The LI-COR 4300 DNA Analyzer is a laboratory instrument designed for DNA sequencing and fragment analysis. It utilizes fluorescent dye-labeled DNA samples and a detection system to identify DNA sequences and fragment sizes.
Automatically generated - may contain errors
Lab products found in correlation
Bigdye terminator cycle sequencing kit, by Thermo Fisher Scientific (2 mentions)
Dneasy plant mini kit, by Qiagen (2 mentions)
Taq polymerase, by Thermo Fisher Scientific (2 mentions)
Irdye 700, by LI COR (2 mentions)
Irdye 800 dye, by LI COR (2 mentions)
Genemapper software, by Thermo Fisher Scientific (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Sequagel xr, by National Diagnostics (1 mentions)
Icycler, by Bio-Rad (1 mentions)
Gentra puregene tissue kit, by Qiagen (1 mentions)
Amplitaq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Sagagt software, by LI COR (1 mentions)
Kasp assay, by LGC (1 mentions)
27 protocols using 4300 dna analyzer
1
Preparation of Fluorescent Tetrahymena Ribozyme
2
Genomic DNA Sequencing for SNP Identification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Total genomic DNA was isolated from the fresh leaves of 435 unrelated individuals, using the DNeasy Plant Mini Kit (Qiagen China). To identify SNPs, the full-length genomic DNA of candidate genes was sequenced using gene-specific primers, which were designed based on the cDNA of candidate genes. The PCR amplification procedure has been described previously (Zhang et al., 2011 (link)). The BigDye Terminator Cycle Sequencing kit (version 3.1, Applied Biosystems) and the 4300 DNA Analyzer (Li-Cor Biosciences, Lincoln, NE, USA) were applied for sequencing. All 129 sequences of the three candidate genes were deposited in NCBI (https://www.ncbi.nlm.nih.gov/ ) under the accession numbers MG873890 –MG873932 for Pto-MIR167a, MG873933–MG873975 for Pto-ARF8, and MG873976–MG874018 for ARFRL. Sequence alignment and SNP identification were conducted by MEGA 5.0 software (Tamura et al., 2011 (link)). Using the Beckman Coulter (Franklin Lakes, NJ, USA) sequencing system, the common SNPs (minor allele frequency [MAF] ≥ 5%) were genotyped across all 435 individuals in the association population (Table S5 ).
3
Microsatellite Marker Development and Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Development of the SSR primers (Table 3 ), Psol 3, Psol 6, and Psol 10 was described elsewhere [35 (link)]. Polymerase chain reactions (PCRs) were performed in 20 μL reaction mixtures, containing 10 mM Tris-HCl and 50 mM KCl buffer, 20–50 ng of total DNA template, 2 mM MgCl2, dNTPs at 0.25 μM, bovine serum albumin (BSA) at 0.5 μg.μL−1, forward primer (F) at 0.16 μM, reverse primer (R) at 0.2 μM, M13 primer IR700 or IR800 at 0.3 μM, and 1 U of Taq DNA polymerase. One of the primers of each of the six pairs contained a fluorophore (IRDye-700 or IRDye-800) for capillary electrophoretic detection. For PCR amplification, the following program of temperatures was used: 5 min at 94°C, 10 cycles of 1 min at 94°C, 1 min at 58°C, decreasing 1°C at each following cycle, 1 min at 72°C, 30 cycles of 40 sec at 94°C, 40 sec at 48°C, 1 min at 72°C, and a final extension of 10 min at 72°C [31 ].
PCR products were analyzed using a 4300 DNA Analyzer (LI-COR, Lincoln, Nebraska, USA). Allele sizes were obtained by comparison with 50–700 bp standards (IRDye 700). Banding pattern analyses were performed using the GeneMapper software (Applied Biosystems) [26 ].
PCR products were analyzed using a 4300 DNA Analyzer (LI-COR, Lincoln, Nebraska, USA). Allele sizes were obtained by comparison with 50–700 bp standards (IRDye 700). Banding pattern analyses were performed using the GeneMapper software (Applied Biosystems) [26 ].
4
Genomic DNA Sequencing and Annotation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Using the DNeasy Plant Mini kit (Qiagen China, Shanghai), total genomic DNA was extracted from fresh young leaves. For sequencing the full-length genomic DNA sequences, specific primers were designed based on each cDNA sequence. PCR amplification was performed according to Zhang et al.22 (link) All the genomic DNA of candidate genes were obtained by direct sequencing of both strands, using conserved T7 and SP6 primers in the BigDye Terminator Cycle Sequencing Kit (version 3.1; Applied Biosystems, Foster City, CA, USA) and a 4300 DNA Analyzer (Li-Cor Biosciences, Lincoln, NE, USA). Full-length genomic DNA sequences of all 11 candidate genes were isolated by PCR amplification from the P. tomentosa LM50 clone (Supplementary Table S2 ). Of these, five of these genes were previously reported in single-marker association studies (Supplementary Table S2 ). Also, these 11 full-length gene sequences were deposited in GenBank (accession numbers shown in Supplementary Table S2 ).
5
Identifying DNA Lesions via Fluorescence Labeling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The IRDye® 700/800 fluorescence-labelled products (2 µl) were denaturated with formamide loading buffer (3 µl) at 95 °C for 5 min (final volume 5 µl), and, after 10 min on ice, denaturated labelled products (1 µl) were subjected to electrophoresis on a polyacrylamide sequencing gel, 80.0% acrylamide, 10% bis-acrylamide/urea and run in the LI-COR 4300 DNA analyzer at 65 °C for 7 h. DNA lesions were identified as the locations in which the presence of Fpg and EndoIII-sensitive sites stopped the Vent exo- from progressing and resulted in an intense dark band. The relative band intensity (RI), quantificated by ImageQuant, were calculated by following formula RI = Ij/Imax, where Ij is the intensity in pixels of each band after background subtraction and Imax is the mean intensity in pixels of the highest intensity bands.24 (link) The RI values were corrected across experiments based on the recovery of the reference standard. The exact position of each base was determined by including labelled DNA fragments obtained by sequencing of the region of interest according to Ruano and Kidd25 (link) and appropriate size IRDye® 700/800 sizing standards markers in the sequencing gel.
6
Genotyping Rat Vkorc1 Variant
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA of a tissue sample from the tail of dead rats mostly collected during or after the treatment was isolated using the DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) and included in a high-resolution melting polymerase chain reaction (HRM-PCR), which amplifies a 141 bp fragment of the Exon 3 of the vkorc1 gene, using the PikoReal Real-Time PCR system (Biozym Scientific GmbH, Hessisch Oldendorf, Germany) (Diaz and Kohn 2020 (link)). Comparing the melting curves, variations at position 139 are detected and assigned as wildtype, heterozygote, or homozygote. In addition, randomly selected amplification products were sequenced to confirm the assignment using a 4300 DNA Analyzer (LI-COR Biosciences, Lincoln, NE, USA [36].
7
Primer Design and PCR Protocol for SSR Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For designing the primer pairs from the flanking sequences of identified SSRs, Primer3 software90 (link),91 (link) was used with the parameters as follows: primer length of 18–27 nucleotides, melting temperatures of 55–65 °C, GC content of 30–70%, and predicted PCR products of 100–300 bp in length. The primer pairs were later controlled for possible duplication of sequences in the genome using IGB software.
The PCR reactions were performed using the M13 tailing PCR procedure92 (link). The forward primers were tailed by adding an M13 sequence labeled with IRDye to the 5′ end. The following PCR protocol was applied: 95 °C initial denaturation for 5 min, 30 cycles at 95 °C for 30 s, annealing temperature 60 °C for 30 s, 72 °C for 1 min, followed by 9 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and then a final extension of 10 min at 72 °C. PCR products were loaded onto 8% denatured polyacrylamide gel and separated by 4300 DNA analyzer (LI-COR, Inc., Lincoln, Nebraska, USA). 1 kb size marker was used to score markers as 1 or 0 for the presence and absence of alleles.
The PCR reactions were performed using the M13 tailing PCR procedure92 (link). The forward primers were tailed by adding an M13 sequence labeled with IRDye to the 5′ end. The following PCR protocol was applied: 95 °C initial denaturation for 5 min, 30 cycles at 95 °C for 30 s, annealing temperature 60 °C for 30 s, 72 °C for 1 min, followed by 9 cycles of 95 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and then a final extension of 10 min at 72 °C. PCR products were loaded onto 8% denatured polyacrylamide gel and separated by 4300 DNA analyzer (LI-COR, Inc., Lincoln, Nebraska, USA). 1 kb size marker was used to score markers as 1 or 0 for the presence and absence of alleles.
8
Transposon Display in Peanut Accessions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Transposon display was conducted as described (Casa et al. 2000 (link)). In brief, 150-ng genomic DNA from each of ten peanut accessions was digested with MseI at 37 °C for 2 h, and the digested DNAs was mixed with the adapters at 4 °C overnight. Preselective amplification was performed using the adaptor primer (5′-GATGAGTCCTGAGTAA-3′) and the AdLINE3 specific primer (5′-GAAGACCTAAGAAGACCATC-3′). PCR reaction for selective amplification was conducted using the adapter primer and AdLINE3 primer (5′-GAAGACCTAAGAAGACCATC-3′, where N was A, T, C, or G). A 6.5% polyacrylamide gel was prerun at 1500 V for 20 min on a LI-COR 4300 DNA Analyzer. Samples (0.5 µl each) were loaded onto a gel and then run for 3.5 h at 1500 V. Image was viewed with both the 700 and 800 channels.
9
Genetic diversity in Pacific and Indian Ocean populations
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
One hundred individuals (data given from Table
1 ) collected from 14 populations in the Pacific and the Indian Ocean were used for the analysis. Details of sample size, names of locations and coordinates are presented in Table
1 . Among 10 primer pairs suggested by Xu et al.
[70 (link)], we used five primer pairs resulting in highly polymorphic bands (HO5, HO8, HO36, HO48 and HO51) (Table
7 ) for PCR. Thirty ng of template DNA was used in each 15 μl PCR including 1x Williams buffer, 0.2 mM dNTPs, 1 U Taq polymerase (MBI Fermentas), and 1 pmol primer each. The PCR was performed in a PTC 200 thermocycler (Biozym-Diagnostik GmbH under the following conditions: initial denaturation for 5 min at 94°C followed by 25 cycles of denaturation for 30 s at 94°C, primer annealing for 30 s at 52 to 59°C and extension for 35 s at 72°C, and terminated by a final hold at 10°C. To each sample, 200 μl of dye (98% formamide, 10 mM EDTA, 0.05% pararosaniline) was added. Reactions were heated up to 72°C for 5 min before loading onto 6% AFLP gels (Sequagel XR, National Diagnostics, Hull, England). For running an AFLP gel on the 4300 DNA Analyzer (LI-COR, Biosciences, Germany) manufacture’s instruction were followed. Base pair lengths obtained from visual analysis was resolved with previously published allele lengths
[70 (link)] and sequencing was performed when necessary.
[70 (link)], we used five primer pairs resulting in highly polymorphic bands (HO5, HO8, HO36, HO48 and HO51) (Table
[70 (link)] and sequencing was performed when necessary.
10
Genetic Diversity Analysis of Turkish Watermelon
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
In this study, genotypes collected from different regions of Turkey by Çukurova University Faculty of Agriculture, Department of Horticulture and purified up to S4-S6 grade were used. Among 250 watermelon lines with good agronomic characteristics, 96 genotypes selected from the previous project were used. These genotypes were selected from among those that were found to be the most genetically different from each other. Some of these genotypes are commercial varieties (35 Sugar baby, 235 Charleston Gray Seminis USA, 238 Dixilee North caroline USA, 365 China, G11 DIMA 4B Hungary and G12 Gyulavari Hungary). Other genotypes originate from Turkey.
DNA isolations were made, and PCR studies were carried out. Equal amounts of formamide loading buffer containing 10 mM EDTA (pH 8.0), 95% formamide, 0.025% bromophenol blue and 0.025% xylene cyanol were added to each tube containing the amplification product. PCR products were loaded on a 30% polyacrylamide gel (Long Ranger, FMC Biozym, Hessisch Oldendorf, Germany) and visualized on the 4300 DNA Analyzer (Li-Cor). M13 reverse (GGATAACAATTTCACACGG) or M13 forward (CACGACGTTGTAAAACGAC) primers were added to the 5' end of the synthetically prepared SSR Forward primers (700 or 800 nm wavelength). Data were analyzed using NTSYS program, UPGMA dendrogram was produced and PCA analyses were performed.
DNA isolations were made, and PCR studies were carried out. Equal amounts of formamide loading buffer containing 10 mM EDTA (pH 8.0), 95% formamide, 0.025% bromophenol blue and 0.025% xylene cyanol were added to each tube containing the amplification product. PCR products were loaded on a 30% polyacrylamide gel (Long Ranger, FMC Biozym, Hessisch Oldendorf, Germany) and visualized on the 4300 DNA Analyzer (Li-Cor). M13 reverse (GGATAACAATTTCACACGG) or M13 forward (CACGACGTTGTAAAACGAC) primers were added to the 5' end of the synthetically prepared SSR Forward primers (700 or 800 nm wavelength). Data were analyzed using NTSYS program, UPGMA dendrogram was produced and PCA analyses were performed.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!