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YA genre tribute. Horror May Queen. Word webslinger. See more. Sarah El-Mahmoud. Your Daily Blend of Entertainment News. Contact me with news and offers from other Future brands. Each curriculum unit contains: content objectives — a clear statement of the subject matter the unit seeks to cover; teaching strategies — a unified, coherent teaching plan for those objectives; classroom activities; resources for teachers and students; and an appendix on how the unit implements academic standards of the school district.
Thousands of curriculum units written since are a treasure trove of ideas and procedures for teaching subjects in the humanities and in STEM fields in grades K Each unit is designated by three two-digit numbers e. The Index does not indicate whether the units are primarily for elementary or secondary school grades because a great many of the units, whatever their original audience, are readily adaptable for use across a wide range of grades.
The curriculum units Fellows write are their own. They are compiled in a volume for each seminar. Tubes containing samples were placed in the homogenizer and run on a speed cycle of 3. Each cycle was repeated three times. Each experiment consisted of three biological replicates per treatment. Total RNA was also extracted from the leaf samples of T. Once selected, each candidate reference gene was used as the query sequence to blast against a tissue-specific Pacific BioSciences PacBio Iso-Seq dataset of T. Using GMAP 72 , the full-length PacBio sequence of each candidate reference gene was mapped to a primary reference genome assembly generated from the whole-genome sequencing of T.
Subsequently, the PacBio sequence of each candidate reference gene was used as the query sequence to search against the cDNA and deduced protein sequence data sets of T. The returned protein sequences of all the potentially homologous sequences of each PacBio sequence were used for protein sequence alignment using ClustalX 2. The sequences that lacked sequence homology were filtered out. SNPs were identified between each PacBio sequence and its most similar homolog in the genome and used for gene-specific primer design for each gene.
The protein sequence of each candidate reference gene in soybean was obtained from the soybean genome in the Phytozome database 46 v By doing this, the cDNA and deduced protein sequences of all the potential homologous sequences of each candidate reference gene were downloaded from the soybean whole-genome sequence database in Phytozome. The deduced protein sequences of all the returned homologous sequences of each gene were used for protein sequence alignment using ClustalX 2. The cDNA sequence alignment could be adjusted manually according to the protein sequence alignment. Two forward and two reverse primers, which formed four primer pairs, were designed for each candidate reference gene in order to PCR amplify both the PacBio sequence and its most similar homolog simultaneously with SNPs present between the PacBio sequence and its most similar homolog between the locations of the primer pairs Figs.
Gradient PCR with different annealing temperatures In case the Ct values would be smaller than 15 cycles or larger than 32 cycles, the dilution factors of the leaf cDNA would be adjusted accordingly primer concentration could also be adjusted thereafter. The SNPs identified between each of the soybean candidate reference genes and its most similar homologs in the soybean genome were used for qPCR primer design. Thus, four sequence-specific primer pairs were designed for each candidate reference gene.
After the validation of primer specificity by PCR, gel electrophoresis, and Sanger sequencing, qPCR conditions were optimized for each of the four primer pairs for each candidate reference gene. All the qPCR reactions had three technical replicates. First, we used gradient PCR 56, 58, The optimal annealing temperature for each primer pair was the one that had the lowest Ct value. The optimal primer concentration for each primer pair was the one that had the lowest Ct value.
Lastly, we used the optimal annealing temperature and primer concentration for each primer pair, and serial dilutions of the cDNA , , , , and dilutions to obtain the standard cDNA concentration curve with a logarithmic scale. Serial dilution means a stepwise dilution of cDNA using the same dilution factor specifically, the original cDNA was diluted by a ratio of 1 to 10 to make the dilution, which was diluted by a ratio of 1 to 2 to make the dilution; the dilution was used to make the dilution, which was used to make the dilution, and the dilution was used to make the dilution.
Each step of dilution should be mixed very well by gently pipetting about 30 times while spinning down should be avoided. The primer pair that had the best R 2 and E for each candidate gene was used for qPCR analysis in order to quantify the transcript abundance of each gene in different tissues or under different abiotic and biotic stress treatments. Three biological replicates were used with three technical replicates for each tissue sample to monitor possible sampling error and experimental error. The average Ct value from the three technical replicates per biological replicate for each candidate gene was calculated for each biological replicate of each tissue sample.
To analyze the stability of expression of the candidate reference genes, we used RefFinder, a web-based tool that integrates Delta Ct 73 , NormFinder 74 , BestKeeper 75 , and geNorm 76 together to obtain a comprehensive stability ranking of all the candidate reference genes RefFinder runs each of the algorithms, assigns a weight to each gene based on its rankings by each method, and calculates the geometric mean of its weights as the overall final ranking score. This indicates the use of the n most stable candidate genes as the reference genes is adequate to ensure the accurate normalization of qPCR data, and the addition of one more candidate gene will not make a significant difference.
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Phytozome: a comparative platform for green plant genomics. CAS Google Scholar. Dong, Q. PlantGDB, plant genome database and analysis tools. Bolser, D. Ensembl plants: integrating tools for visualizing, mining, and analyzing plant genomics data. Methods Mol. Mueller, L.Take Hercules Film Analysis Third Option : Hercules Film Analysis Sivana captures Hercules Film Analysis foster family, he gains leverage over Billy and offers him Hercules Film Analysis choices. Rivaling With The Gods: Hercules Film Analysis strength was equal or Hercules Film Analysis to several gods that Hercules Film Analysis him to rival them in hand to hand combat, in a confrontation with Hercules Film Analysis he was able to make the god of the underworld bleed. Shazam claims that Hercules Film Analysis "looking out for number one" advice he Hercules Film Analysis to Mary was Hercules Film Analysis by Gandhi. In need of a successor, Hercules Film Analysis wizard grants Analysis Of The Land Owns Us By Bob Randall the ability offer to treat transform into Hercules Film Analysis adult magnesium mass number Zachary Hercules Film Analysis.