CRISPR.pptx
•Download as PPTX, PDF•
0 likes•43 views
This document provides requirements and methods for generating a recombinant vector containing two tandem guide RNAs (gRNAs) targeting the dLarp7 gene in Drosophila. The requirements include primer sets for amplifying the gRNA sequences, vector backbone, and detecting the recombinant vector, as well as Gibson Assembly reagents. The method describes generating the recombinant pCDF4 vector containing the two gRNAs, microinjecting it into flies containing an attP docking site, crossing these flies with Cas9 flies to induce deletion of the dLarp7 gene, and screening F2 progeny by PCR to detect the deletion.
Report
Share
Report
Share
Recommended
Molecular markers and plant breeding4
This document discusses the techniques of restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR). RFLP involves using restriction enzymes to cut DNA into fragments, which are then analyzed via gel electrophoresis and probing. PCR, developed in 1985, allows for exponential amplification of specific DNA regions using oligonucleotide primers, DNA polymerase, and thermal cycling. The PCR process involves repeated cycles of template DNA denaturation, primer annealing, and fragment extension, resulting in millions of copies of the target DNA. Both RFLP and PCR are useful for analyzing DNA samples.
Lec 6
This document summarizes several types of molecular marker techniques used in PCR-based DNA fingerprinting: Random Amplified Polymorphic DNA (RAPD), Sequence-tagged Sites (STS), Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeats (SSR), Inter-Simple Sequence Repeats (ISSR), Sequence Characterized Amplified Regions (SCAR), and Cleaved Amplified Polymorphic Sequences (CAPS). It provides brief descriptions of each technique, including what type of DNA they use, how polymorphisms are detected, common primers and enzymes used, and some of their advantages and limitations.
Mock Qualifying Exam
This study aims to identify the corepressor of daf-3 that mediates dauer formation in C. elegans in response to high temperatures, and to determine the mechanism by which daf-3 switches cofactors between repressor and activator in different temperatures. The specific aims are to first identify the daf-3 corepressor using RNAi, deletion mutants, protein extracts, and mass spectrometry. The second aim is to study the temperature dependent expression of daf-5 and the corepressor, their competitive binding to daf-3, and changes in daf-3 mRNA isoforms, to understand how daf-3 switches cofactors between repressor and activator in response to temperature
Q biomarkercn
This document describes qBiomarker Copy Number PCR Assays and Arrays, which are tools for measuring copy number variations and alterations in DNA. The assays include primer sets targeting loci known to be amplified or deleted. They are available in plate formats to simultaneously analyze up to 95 loci under uniform cycling conditions. The assays provide focused profiling of copy number changes for various diseases and pathways.
Biochain PCR Products
The document discusses BioChain's products for PCR and sample preparation, including PCR enzymes, reverse transcriptases, master mixes, dNTPs, and supporting reagents. It provides details on BioChain's Taq DNA polymerase and Hot Start Taq DNA polymerase, which are produced under strict quality control. It also describes BioChain's UltraScript reverse transcriptase, which is ideal for cDNA synthesis of templates with secondary structure or high GC content. Furthermore, it mentions BioChain's pre-mixed master mixes for standard and quantitative PCR, which offer convenience and reproducibility.
Scfv phage library
This document discusses methods for constructing antibody scFv fragment libraries displayed on phage (phage display scFv libraries) for stability selection. It describes several strategies for PCR amplification and assembly of variable heavy (VH) and light (VL) chain domains connected by a linker to produce single-chain Fv fragments (scFvs). These include normal PCR, pull-through PCR, overlap extension PCR, and RACE PCR strategies. It then provides a basic protocol for constructing naive scFv libraries using peripheral blood mononuclear cells, including steps for VH and VL repertoire construction and assembly into a phagemid vector for phage display.
Cpf1-based genome editing using ribonucleoprotein complexes
The CRISPR-Cpf1 nuclease is the best alternative to the commonly used Cas9 for genome editing. Cpf1 recognizes a protospacer adjacent motif (PAM) sequence of TTTV, which differs from the Cas9 PAM sequence, NGG. Having Cpf1 as a second option increases the likelihood of editing as close as possible to your desired target site. In this presentation, Dr Rolf Turk introduces the optimized Alt-R™ CRISPR-Cpf1 System and explains how it can be used as a powerful new tool for your genome editing research. Dr Turk presents the basics of the system, as well as protocols for getting started in genome editing.
NGS Data Preprocessing
This document discusses next generation sequencing (NGS) data preprocessing and quality control. It provides a brief history of DNA sequencing technologies and compares current NGS platforms. The importance of quality control and preprocessing NGS data is explained. Key metrics for assessing NGS data quality are described, including per base quality scores, GC content distributions, and k-mer content. Tools for preprocessing (Fastx-toolkit) and quality control (FastQC) are introduced.
Recommended
Molecular markers and plant breeding4
This document discusses the techniques of restriction fragment length polymorphism (RFLP) and polymerase chain reaction (PCR). RFLP involves using restriction enzymes to cut DNA into fragments, which are then analyzed via gel electrophoresis and probing. PCR, developed in 1985, allows for exponential amplification of specific DNA regions using oligonucleotide primers, DNA polymerase, and thermal cycling. The PCR process involves repeated cycles of template DNA denaturation, primer annealing, and fragment extension, resulting in millions of copies of the target DNA. Both RFLP and PCR are useful for analyzing DNA samples.
Lec 6
This document summarizes several types of molecular marker techniques used in PCR-based DNA fingerprinting: Random Amplified Polymorphic DNA (RAPD), Sequence-tagged Sites (STS), Amplified Fragment Length Polymorphism (AFLP), Simple Sequence Repeats (SSR), Inter-Simple Sequence Repeats (ISSR), Sequence Characterized Amplified Regions (SCAR), and Cleaved Amplified Polymorphic Sequences (CAPS). It provides brief descriptions of each technique, including what type of DNA they use, how polymorphisms are detected, common primers and enzymes used, and some of their advantages and limitations.
Mock Qualifying Exam
This study aims to identify the corepressor of daf-3 that mediates dauer formation in C. elegans in response to high temperatures, and to determine the mechanism by which daf-3 switches cofactors between repressor and activator in different temperatures. The specific aims are to first identify the daf-3 corepressor using RNAi, deletion mutants, protein extracts, and mass spectrometry. The second aim is to study the temperature dependent expression of daf-5 and the corepressor, their competitive binding to daf-3, and changes in daf-3 mRNA isoforms, to understand how daf-3 switches cofactors between repressor and activator in response to temperature
Q biomarkercn
This document describes qBiomarker Copy Number PCR Assays and Arrays, which are tools for measuring copy number variations and alterations in DNA. The assays include primer sets targeting loci known to be amplified or deleted. They are available in plate formats to simultaneously analyze up to 95 loci under uniform cycling conditions. The assays provide focused profiling of copy number changes for various diseases and pathways.
Biochain PCR Products
The document discusses BioChain's products for PCR and sample preparation, including PCR enzymes, reverse transcriptases, master mixes, dNTPs, and supporting reagents. It provides details on BioChain's Taq DNA polymerase and Hot Start Taq DNA polymerase, which are produced under strict quality control. It also describes BioChain's UltraScript reverse transcriptase, which is ideal for cDNA synthesis of templates with secondary structure or high GC content. Furthermore, it mentions BioChain's pre-mixed master mixes for standard and quantitative PCR, which offer convenience and reproducibility.
Scfv phage library
This document discusses methods for constructing antibody scFv fragment libraries displayed on phage (phage display scFv libraries) for stability selection. It describes several strategies for PCR amplification and assembly of variable heavy (VH) and light (VL) chain domains connected by a linker to produce single-chain Fv fragments (scFvs). These include normal PCR, pull-through PCR, overlap extension PCR, and RACE PCR strategies. It then provides a basic protocol for constructing naive scFv libraries using peripheral blood mononuclear cells, including steps for VH and VL repertoire construction and assembly into a phagemid vector for phage display.
Cpf1-based genome editing using ribonucleoprotein complexes
The CRISPR-Cpf1 nuclease is the best alternative to the commonly used Cas9 for genome editing. Cpf1 recognizes a protospacer adjacent motif (PAM) sequence of TTTV, which differs from the Cas9 PAM sequence, NGG. Having Cpf1 as a second option increases the likelihood of editing as close as possible to your desired target site. In this presentation, Dr Rolf Turk introduces the optimized Alt-R™ CRISPR-Cpf1 System and explains how it can be used as a powerful new tool for your genome editing research. Dr Turk presents the basics of the system, as well as protocols for getting started in genome editing.
NGS Data Preprocessing
This document discusses next generation sequencing (NGS) data preprocessing and quality control. It provides a brief history of DNA sequencing technologies and compares current NGS platforms. The importance of quality control and preprocessing NGS data is explained. Key metrics for assessing NGS data quality are described, including per base quality scores, GC content distributions, and k-mer content. Tools for preprocessing (Fastx-toolkit) and quality control (FastQC) are introduced.
Log Visualization - Bellua BCS 2006
This document discusses visualizing logfiles using graphs. It begins with an introduction on how graphs can help detect both expected and unexpected events while reducing analysis and response times. It then covers graphing basics like how to generate a graph by parsing a logfile and normalizing the data. Different types of visual graphs are presented, including link graphs and tree maps. Link graph configurations using different node types like source IP, name, destination IP are demonstrated. Tree maps can organize data hierarchically by protocol and service to visualize network traffic proportions.
Enzymes In Transcription, Translation and Replication
I have entered here all the enzymes related to the three most important function in Molecular Biology - Replication Transcription and translation .They are concised in three simple charts. Also diagrams of 5 types of Gene that code for mRNA,rRNA,tRNA that are Transcribed by different RNA polymerase.
Target capture of DNA from FFPE samples— recommendations for generating robus...
Target capture of DNA from FFPE samples— recommendations for generating robus...Integrated DNA Technologies
Cancer therapies that target specific pathways can be more effective than established, nonspecific chemotherapy and radiation treatments, and may prevent side effects on healthy tissues. Such targeted therapies can only be applied after underlying gene mutations have been identified. However, detecting low frequency variants from clinically relevant samples poses significant challenges. Specimens are routinely formalin-fixed and paraffin-embedded (FFPE) for histology, which can decrease the efficiency of NGS library preparation. In this presentation, we discuss approaches for extraction of DNA from FFPE samples, and recommend quality control assays to guide parameter selection for library construction and sequencing depth. Reducing off-target events in CRISPR genome editing applications with a novel...
Reducing off-target events in CRISPR genome editing applications with a novel...Integrated DNA Technologies
The CRISPR-Cas9 system demonstrates unparalleled genome editing efficiency in a broad range of species and cell types, but it suffers from concerns related to target specificity. Modified guide RNAs and mutant Cas9 proteins have been developed to reduce off-target editing but, in many cases, the alterations also significantly reduce on-target editing performance. In this presentation, Dr Chris Vakulskas discusses a novel, high-fidelity Cas9 protein that reduces off-target gene editing, while maintaining high on-target activity. Dr Vakulskas presents data from the development of the new Alt-R® S.p. HiFi Cas9 Nuclease 3NLS and describes its usefulness in mitigating unwanted off-target gene editing, without the issues associated with transfection of plasmid DNA.Lectut btn-202-ppt-l29. applications of pcr-i (1)
PCR can be used for a variety of applications including isolating and cloning genes, cDNA synthesis, mutagenesis, molecular markers, analyzing ancient DNA, medical diagnostics, and forensics. Molecular biologists use PCR to identify specific genes and make multiple copies. Forensic technicians use PCR to help identify suspects and victims based on DNA amplification patterns. PCR has impacted many areas of life and promises further exciting discoveries.
Increasing genome editing efficiency with optimized CRISPR-Cas enzymes
Genome editing by CRISPR systems has proven to be groundbreaking for basic biomedical research with significant implications for the treatment of human diseases. While the CRISPR-Cas9 and CRISPR-Cas12a (Cpf1) systems enable genome editing in a broad range of host species and cell types, both can exhibit poor editing efficiencies at specific target sites or in systems where delivery of CRISPR reagents is difficult. There are concerns about target specificity of the CRISPR-Cas9 system and, in many cases, typical remedies such as modified guide RNAs or mutant Cas9 proteins cause loss of genome editing efficiency. Many of these solutions for improving specificity were developed for delivery of the Cas9-gRNA complex via plasmid DNA vectors rather than delivery as ribonucleoproteins (RNPs). However, RNP delivery of CRISPR reagents is being increasingly used because of the risk of unwanted stimulation of the immune system by plasmid delivery.
In this webinar, Dr Vakulskas discusses improved Cas9 and Cas12a (Cpf1) nucleases that have been optimized to significantly increase editing efficiency in living cells. He also presents data showing that IDT’s latest high-fidelity Cas9, Alt-R HiFi S.p. Cas9 V3, increases on-target editing efficiency and dramatically reduces off-target editing.
Real time pcr market & end user needs survey
Parioforma Ltd. is a London-based business consultancy that provides market research services. This report summarizes a study on quantitative real-time PCR (qRT-PCR) technology and market trends. Key findings include that qRT-PCR is a mature technique seeing growth in automation, high-throughput applications, and standardized assays. While instruments are reliable, future improvements may focus more on sample preparation and data analysis to support the large amounts of data generated.
Knockdown of lncRNAs: exploring RNAi and antisense oligo methods
Thousands of different long non-coding RNAs (lncRNAs) exist in mammalian cells. lncRNAs do not encode proteins but can be very important for cell function. Studying their functions can be difficult because of their diverse modes of action. One method to discern cellular function is by selective knockdown of a specific lncRNA species. However, achieving consistent knockdown has proven to be more challenging for lncRNAs than for mRNAs or miRNAs. In this presentation, we discuss some of the issues encountered with lncRNA research. We cover antisense oligonucleotide (ASO) and small interfering RNA (siRNA) methods for lncRNA knockdown. And, we show how cellular localization of a specific lncRNA target informs the choice of knockdown method.
Anne_Vaittinen_advanced_seminar_presentation
This document summarizes different RNA sequencing library preparation methods and their results. It describes using Nextera, TotalScript RNA-Seq Kit, NEXTflex Directional RNA-Seq Kit, ScriptSeq v2 RNA-Seq, and NEBNext Ultra Directional RNA Library Prep Kit to prepare RNA libraries. It then details the results of quality control checks, sequencing output, alignment rates, and levels of antisense transcription detected for each method, finding that the ScriptSeq v2 RNAseq kit was the most suitable.
Real time PCR
Real time PCR allows for monitoring of DNA amplification during polymerase chain reaction (PCR), rather than just at the end. There are two main detection methods: using non-specific fluorescent dyes that bind to double stranded DNA, and using sequence-specific fluorescent probes. Common non-specific dyes include SYBR Green I, while TaqMan probes are an example of sequence-specific probes that use fluorescence resonance energy transfer. Real time PCR has applications in disease diagnosis, microbiology research on food and water safety, and quantifying gene expression levels.
20140711 3 t_clark_ercc2.0_workshop
This document discusses PacBio single molecule real-time (SMRT) sequencing of full-length cDNA transcripts. It summarizes the current challenges with transcript assembly using short-read sequencing and describes how PacBio Iso-Seq provides high-quality, full-length transcript isoforms through single-molecule long-read sequencing of cDNA. The document also reviews size selection methods like SageELF that can separate transcripts into different size fractions for sequencing.
Bioproject2
The document describes the process of protein synthesis, which has two main steps: transcription and translation. During transcription, RNA polymerase binds to DNA and builds an mRNA strand using the coding region as a template, before the mRNA strand exits the nucleus. Translation then occurs on ribosomes, where tRNAs bring amino acids to the mRNA start codon and assemble a protein based on the mRNA sequence until reaching the stop codon.
Optimized methods to use Cas9 nickases in genome editing
The CRISPR-Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. One of the key components of this editing system is Cas9 endonuclease. The cleavage activity of the S. pyogenes Cas9 enzyme is mediated by the coordinated functions of two catalytic domains and creates blunt-ended, double-stranded breaks. Alanine substitution at key residues within these domains creates two Cas9 nickase variants. Variant D10A produces a nick on the targeting strand, while H840A nicks the non-targeting strand. This double nicking strategy can be leveraged to reduce unwanted off-target effect. However, the nickase experiments can be inherently more complicated than standard CRISPR-Cas9 editing, given the requirement for two guide RNAs to function simultaneously.
In this webinar, both Shuqi Yan and Mollie Schubert present the data from the characterization of a number of factors that impact the efficiency of cooperative nicking in cell cultures. They also summarize a few key design considerations for achieving efficient gene disruption or homology directed repair (HDR) when planning your nickase experiments.
Learn more: http://www.idtdna.com/pages/products/crispr-genome-editing
Increase efficiency of genome editing with the Alt-R™ CRISPR-Cas9 System: Des...
Increase efficiency of genome editing with the Alt-R™ CRISPR-Cas9 System: Des...Integrated DNA Technologies
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. In a recent webinar, "New RNA Tools for Optimized CRISPR/Cas 9 Genome Editing", we presented how we developed the Alt-R™ CRISPR-Cas9 System for genome editing. Here, we take a look at designing your target sequences and ordering them as Alt-R CRISPR crRNA. We review the other components of the system and walk through the experimental process step by step, from design to evaluation of editing potency. We also discuss challenges and potential pitfalls and provide tips and guidance towards successful genome editing experiments. Learn more: http://www.idtdna.com/crispr20140711 4 e_tseng_ercc2.0_workshop
This document discusses technical variability in PacBio full-length cDNA sequencing (Iso-Seq). It summarizes the Iso-Seq experimental and informatics pipelines, and analyzes read count variation between technical replicates and tissues. While technical variation is minimal, amplification biases from different enzymes and detection limits remain areas for improvement. Combining Iso-Seq with short-read data may help address these challenges.
Genome Assembly
The document provides an overview of plant genome sequence assembly, including:
1) A brief history of sequencing technologies and their improvements over time, from Sanger sequencing to newer technologies producing longer reads.
2) Key steps in a sequencing project including read processing, filtering, and corrections before assembly into contigs and scaffolds using appropriate software.
3) Factors to consider for experimental design and assembly optimization such as sequencing depth, library types, and software choices depending on the genome and data characteristics.
Chigot poster2007
The ArrayGradeTM FFPE RNA isolation kit provides a more effective method for isolating RNA from formalin-fixed paraffin-embedded (FFPE) tissue samples. It yields RNA of higher quantity and quality compared to other commercial kits. The RNA isolated has greater compatibility with microarray and real-time PCR applications, providing more positive results and sensitivity. This allows researchers to reliably perform gene expression profiling on archived FFPE samples to gain insights into cancer progression and other disease studies.
RNA polymerase II depletion promotes transcription of alternative mRNA species
Depletion of RNA polymerase II and I, but not III, promotes transcription of alternative mRNA species and induces expression of specific genes in yeast. Chromatin immunoprecipitation experiments showed this was not due to increased occupancy of RNA polymerase at gene loci. Genome-wide analysis found depletion of RNA polymerase II altered polyadenylation site usage for many mRNAs and induced a subset of mRNAs. This suggests transcriptional stress responses modify the transcriptional machinery and gene expression beyond standard mechanisms used for other stresses.
Nested pcr
The document discusses nested PCR, which is a modification of polymerase chain reaction (PCR) that improves specificity. It involves two rounds of PCR where the product of the first reaction is used as a template for the second reaction with a nested primer set. This increases specificity by reducing non-specific binding. Some key advantages are improved accuracy and sensitivity for low abundance targets or difficult templates. However, it is more time-consuming and costly than standard PCR due to the extra reagents and steps required. Nested PCR has applications in microbial detection, genetic analysis, and other areas where high specificity is needed.
Ribonucleoprotein delivery of CRISPR-Cas9 reagents for increased gene editing...
Ribonucleoprotein delivery of CRISPR-Cas9 reagents for increased gene editing...Integrated DNA Technologies
Struggling with low editing efficiency or delivery problems in primary or difficult-to-transfect cells? In this presentation, learn about the advantages of using a Cas9:crRNA:tracrRNA ribonucleoprotein (RNP) complex for genome editing. We show the benefits of using RNP complexes, including ease of use, limiting off-target effects, and stability. We also present data showing how genome editing efficiency rates are improved by our Cas9 electroporation enhancer. Furthermore, we provide advice on how to optimize transfection using the Alt-R™ CRISPR-Cas9 System in combination with different electroporation methodologies.Molecular markers
This document discusses genetic markers used in plant breeding. It defines genetic markers as any phenotypic difference controlled by genes that can be used to study inheritance or select for traits. The document describes two main types of genetic markers - morphological markers which are visible traits, and molecular markers which are detected at the DNA level. It provides details on various molecular marker techniques including RFLP, RAPD, AFLP, SSRs, and SNPs. It explains how each technique works and its advantages and disadvantages. The key information is that genetic markers are tools that allow studying inheritance of genes and selecting plants for breeding based on associated genetic differences.
19_21Translation
This document summarizes key events in translation including:
1. tRNAs act as adaptor molecules that pair each mRNA codon to its corresponding amino acid. Francis Crick first proposed this "adaptor hypothesis".
2. tRNAs undergo processing and modification before being charged with their cognate amino acids by aminoacyl tRNA synthetases.
3. The genetic code was deciphered through experiments using RNA polymers and identification of codon frequencies. Wobble base pairing allows multiple codons to code for the same amino acid.
4. Ribosomes catalyze peptide bond formation using rRNA. Bacterial and eukaryotic ribosomes differ in their subunit composition and initiation factors. Elongation
More Related Content
What's hot
Log Visualization - Bellua BCS 2006
This document discusses visualizing logfiles using graphs. It begins with an introduction on how graphs can help detect both expected and unexpected events while reducing analysis and response times. It then covers graphing basics like how to generate a graph by parsing a logfile and normalizing the data. Different types of visual graphs are presented, including link graphs and tree maps. Link graph configurations using different node types like source IP, name, destination IP are demonstrated. Tree maps can organize data hierarchically by protocol and service to visualize network traffic proportions.
Enzymes In Transcription, Translation and Replication
I have entered here all the enzymes related to the three most important function in Molecular Biology - Replication Transcription and translation .They are concised in three simple charts. Also diagrams of 5 types of Gene that code for mRNA,rRNA,tRNA that are Transcribed by different RNA polymerase.
Target capture of DNA from FFPE samples— recommendations for generating robus...
Target capture of DNA from FFPE samples— recommendations for generating robus...Integrated DNA Technologies
Cancer therapies that target specific pathways can be more effective than established, nonspecific chemotherapy and radiation treatments, and may prevent side effects on healthy tissues. Such targeted therapies can only be applied after underlying gene mutations have been identified. However, detecting low frequency variants from clinically relevant samples poses significant challenges. Specimens are routinely formalin-fixed and paraffin-embedded (FFPE) for histology, which can decrease the efficiency of NGS library preparation. In this presentation, we discuss approaches for extraction of DNA from FFPE samples, and recommend quality control assays to guide parameter selection for library construction and sequencing depth. Reducing off-target events in CRISPR genome editing applications with a novel...
Reducing off-target events in CRISPR genome editing applications with a novel...Integrated DNA Technologies
The CRISPR-Cas9 system demonstrates unparalleled genome editing efficiency in a broad range of species and cell types, but it suffers from concerns related to target specificity. Modified guide RNAs and mutant Cas9 proteins have been developed to reduce off-target editing but, in many cases, the alterations also significantly reduce on-target editing performance. In this presentation, Dr Chris Vakulskas discusses a novel, high-fidelity Cas9 protein that reduces off-target gene editing, while maintaining high on-target activity. Dr Vakulskas presents data from the development of the new Alt-R® S.p. HiFi Cas9 Nuclease 3NLS and describes its usefulness in mitigating unwanted off-target gene editing, without the issues associated with transfection of plasmid DNA.Lectut btn-202-ppt-l29. applications of pcr-i (1)
PCR can be used for a variety of applications including isolating and cloning genes, cDNA synthesis, mutagenesis, molecular markers, analyzing ancient DNA, medical diagnostics, and forensics. Molecular biologists use PCR to identify specific genes and make multiple copies. Forensic technicians use PCR to help identify suspects and victims based on DNA amplification patterns. PCR has impacted many areas of life and promises further exciting discoveries.
Increasing genome editing efficiency with optimized CRISPR-Cas enzymes
Genome editing by CRISPR systems has proven to be groundbreaking for basic biomedical research with significant implications for the treatment of human diseases. While the CRISPR-Cas9 and CRISPR-Cas12a (Cpf1) systems enable genome editing in a broad range of host species and cell types, both can exhibit poor editing efficiencies at specific target sites or in systems where delivery of CRISPR reagents is difficult. There are concerns about target specificity of the CRISPR-Cas9 system and, in many cases, typical remedies such as modified guide RNAs or mutant Cas9 proteins cause loss of genome editing efficiency. Many of these solutions for improving specificity were developed for delivery of the Cas9-gRNA complex via plasmid DNA vectors rather than delivery as ribonucleoproteins (RNPs). However, RNP delivery of CRISPR reagents is being increasingly used because of the risk of unwanted stimulation of the immune system by plasmid delivery.
In this webinar, Dr Vakulskas discusses improved Cas9 and Cas12a (Cpf1) nucleases that have been optimized to significantly increase editing efficiency in living cells. He also presents data showing that IDT’s latest high-fidelity Cas9, Alt-R HiFi S.p. Cas9 V3, increases on-target editing efficiency and dramatically reduces off-target editing.
Real time pcr market & end user needs survey
Parioforma Ltd. is a London-based business consultancy that provides market research services. This report summarizes a study on quantitative real-time PCR (qRT-PCR) technology and market trends. Key findings include that qRT-PCR is a mature technique seeing growth in automation, high-throughput applications, and standardized assays. While instruments are reliable, future improvements may focus more on sample preparation and data analysis to support the large amounts of data generated.
Knockdown of lncRNAs: exploring RNAi and antisense oligo methods
Thousands of different long non-coding RNAs (lncRNAs) exist in mammalian cells. lncRNAs do not encode proteins but can be very important for cell function. Studying their functions can be difficult because of their diverse modes of action. One method to discern cellular function is by selective knockdown of a specific lncRNA species. However, achieving consistent knockdown has proven to be more challenging for lncRNAs than for mRNAs or miRNAs. In this presentation, we discuss some of the issues encountered with lncRNA research. We cover antisense oligonucleotide (ASO) and small interfering RNA (siRNA) methods for lncRNA knockdown. And, we show how cellular localization of a specific lncRNA target informs the choice of knockdown method.
Anne_Vaittinen_advanced_seminar_presentation
This document summarizes different RNA sequencing library preparation methods and their results. It describes using Nextera, TotalScript RNA-Seq Kit, NEXTflex Directional RNA-Seq Kit, ScriptSeq v2 RNA-Seq, and NEBNext Ultra Directional RNA Library Prep Kit to prepare RNA libraries. It then details the results of quality control checks, sequencing output, alignment rates, and levels of antisense transcription detected for each method, finding that the ScriptSeq v2 RNAseq kit was the most suitable.
Real time PCR
Real time PCR allows for monitoring of DNA amplification during polymerase chain reaction (PCR), rather than just at the end. There are two main detection methods: using non-specific fluorescent dyes that bind to double stranded DNA, and using sequence-specific fluorescent probes. Common non-specific dyes include SYBR Green I, while TaqMan probes are an example of sequence-specific probes that use fluorescence resonance energy transfer. Real time PCR has applications in disease diagnosis, microbiology research on food and water safety, and quantifying gene expression levels.
20140711 3 t_clark_ercc2.0_workshop
This document discusses PacBio single molecule real-time (SMRT) sequencing of full-length cDNA transcripts. It summarizes the current challenges with transcript assembly using short-read sequencing and describes how PacBio Iso-Seq provides high-quality, full-length transcript isoforms through single-molecule long-read sequencing of cDNA. The document also reviews size selection methods like SageELF that can separate transcripts into different size fractions for sequencing.
Bioproject2
The document describes the process of protein synthesis, which has two main steps: transcription and translation. During transcription, RNA polymerase binds to DNA and builds an mRNA strand using the coding region as a template, before the mRNA strand exits the nucleus. Translation then occurs on ribosomes, where tRNAs bring amino acids to the mRNA start codon and assemble a protein based on the mRNA sequence until reaching the stop codon.
Optimized methods to use Cas9 nickases in genome editing
The CRISPR-Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. One of the key components of this editing system is Cas9 endonuclease. The cleavage activity of the S. pyogenes Cas9 enzyme is mediated by the coordinated functions of two catalytic domains and creates blunt-ended, double-stranded breaks. Alanine substitution at key residues within these domains creates two Cas9 nickase variants. Variant D10A produces a nick on the targeting strand, while H840A nicks the non-targeting strand. This double nicking strategy can be leveraged to reduce unwanted off-target effect. However, the nickase experiments can be inherently more complicated than standard CRISPR-Cas9 editing, given the requirement for two guide RNAs to function simultaneously.
In this webinar, both Shuqi Yan and Mollie Schubert present the data from the characterization of a number of factors that impact the efficiency of cooperative nicking in cell cultures. They also summarize a few key design considerations for achieving efficient gene disruption or homology directed repair (HDR) when planning your nickase experiments.
Learn more: http://www.idtdna.com/pages/products/crispr-genome-editing
Increase efficiency of genome editing with the Alt-R™ CRISPR-Cas9 System: Des...
Increase efficiency of genome editing with the Alt-R™ CRISPR-Cas9 System: Des...Integrated DNA Technologies
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying genomes of organisms ranging from E. coli to humans. In a recent webinar, "New RNA Tools for Optimized CRISPR/Cas 9 Genome Editing", we presented how we developed the Alt-R™ CRISPR-Cas9 System for genome editing. Here, we take a look at designing your target sequences and ordering them as Alt-R CRISPR crRNA. We review the other components of the system and walk through the experimental process step by step, from design to evaluation of editing potency. We also discuss challenges and potential pitfalls and provide tips and guidance towards successful genome editing experiments. Learn more: http://www.idtdna.com/crispr20140711 4 e_tseng_ercc2.0_workshop
This document discusses technical variability in PacBio full-length cDNA sequencing (Iso-Seq). It summarizes the Iso-Seq experimental and informatics pipelines, and analyzes read count variation between technical replicates and tissues. While technical variation is minimal, amplification biases from different enzymes and detection limits remain areas for improvement. Combining Iso-Seq with short-read data may help address these challenges.
Genome Assembly
The document provides an overview of plant genome sequence assembly, including:
1) A brief history of sequencing technologies and their improvements over time, from Sanger sequencing to newer technologies producing longer reads.
2) Key steps in a sequencing project including read processing, filtering, and corrections before assembly into contigs and scaffolds using appropriate software.
3) Factors to consider for experimental design and assembly optimization such as sequencing depth, library types, and software choices depending on the genome and data characteristics.
Chigot poster2007
The ArrayGradeTM FFPE RNA isolation kit provides a more effective method for isolating RNA from formalin-fixed paraffin-embedded (FFPE) tissue samples. It yields RNA of higher quantity and quality compared to other commercial kits. The RNA isolated has greater compatibility with microarray and real-time PCR applications, providing more positive results and sensitivity. This allows researchers to reliably perform gene expression profiling on archived FFPE samples to gain insights into cancer progression and other disease studies.
RNA polymerase II depletion promotes transcription of alternative mRNA species
Depletion of RNA polymerase II and I, but not III, promotes transcription of alternative mRNA species and induces expression of specific genes in yeast. Chromatin immunoprecipitation experiments showed this was not due to increased occupancy of RNA polymerase at gene loci. Genome-wide analysis found depletion of RNA polymerase II altered polyadenylation site usage for many mRNAs and induced a subset of mRNAs. This suggests transcriptional stress responses modify the transcriptional machinery and gene expression beyond standard mechanisms used for other stresses.
Nested pcr
The document discusses nested PCR, which is a modification of polymerase chain reaction (PCR) that improves specificity. It involves two rounds of PCR where the product of the first reaction is used as a template for the second reaction with a nested primer set. This increases specificity by reducing non-specific binding. Some key advantages are improved accuracy and sensitivity for low abundance targets or difficult templates. However, it is more time-consuming and costly than standard PCR due to the extra reagents and steps required. Nested PCR has applications in microbial detection, genetic analysis, and other areas where high specificity is needed.
Ribonucleoprotein delivery of CRISPR-Cas9 reagents for increased gene editing...
Ribonucleoprotein delivery of CRISPR-Cas9 reagents for increased gene editing...Integrated DNA Technologies
Struggling with low editing efficiency or delivery problems in primary or difficult-to-transfect cells? In this presentation, learn about the advantages of using a Cas9:crRNA:tracrRNA ribonucleoprotein (RNP) complex for genome editing. We show the benefits of using RNP complexes, including ease of use, limiting off-target effects, and stability. We also present data showing how genome editing efficiency rates are improved by our Cas9 electroporation enhancer. Furthermore, we provide advice on how to optimize transfection using the Alt-R™ CRISPR-Cas9 System in combination with different electroporation methodologies.What's hot (20)
Enzymes In Transcription, Translation and Replication
Enzymes In Transcription, Translation and Replication
Target capture of DNA from FFPE samples— recommendations for generating robus...
Target capture of DNA from FFPE samples— recommendations for generating robus...
Reducing off-target events in CRISPR genome editing applications with a novel...
Reducing off-target events in CRISPR genome editing applications with a novel...
Increasing genome editing efficiency with optimized CRISPR-Cas enzymes
Increasing genome editing efficiency with optimized CRISPR-Cas enzymes
Knockdown of lncRNAs: exploring RNAi and antisense oligo methods
Knockdown of lncRNAs: exploring RNAi and antisense oligo methods
Optimized methods to use Cas9 nickases in genome editing
Optimized methods to use Cas9 nickases in genome editing
Increase efficiency of genome editing with the Alt-R™ CRISPR-Cas9 System: Des...
Increase efficiency of genome editing with the Alt-R™ CRISPR-Cas9 System: Des...
RNA polymerase II depletion promotes transcription of alternative mRNA species
RNA polymerase II depletion promotes transcription of alternative mRNA species
Ribonucleoprotein delivery of CRISPR-Cas9 reagents for increased gene editing...
Ribonucleoprotein delivery of CRISPR-Cas9 reagents for increased gene editing...
Similar to CRISPR.pptx
Molecular markers
This document discusses genetic markers used in plant breeding. It defines genetic markers as any phenotypic difference controlled by genes that can be used to study inheritance or select for traits. The document describes two main types of genetic markers - morphological markers which are visible traits, and molecular markers which are detected at the DNA level. It provides details on various molecular marker techniques including RFLP, RAPD, AFLP, SSRs, and SNPs. It explains how each technique works and its advantages and disadvantages. The key information is that genetic markers are tools that allow studying inheritance of genes and selecting plants for breeding based on associated genetic differences.
19_21Translation
This document summarizes key events in translation including:
1. tRNAs act as adaptor molecules that pair each mRNA codon to its corresponding amino acid. Francis Crick first proposed this "adaptor hypothesis".
2. tRNAs undergo processing and modification before being charged with their cognate amino acids by aminoacyl tRNA synthetases.
3. The genetic code was deciphered through experiments using RNA polymers and identification of codon frequencies. Wobble base pairing allows multiple codons to code for the same amino acid.
4. Ribosomes catalyze peptide bond formation using rRNA. Bacterial and eukaryotic ribosomes differ in their subunit composition and initiation factors. Elongation
Flipbook
The document describes the processes of transcription and translation. In transcription, RNA polymerase binds to DNA and unwinds it before creating mRNA using the DNA as a template. The mRNA then leaves the nucleus and enters the cytoplasm. In translation, rRNA forms ribosomes which mRNA binds to. tRNA transfers amino acids based on the mRNA sequence, forming peptide bonds and ultimately a completed protein when a stop codon is reached.
Advenced molecular techniques in molecular medical genetics laboratory
The document discusses various molecular techniques used in genetic testing and molecular medical genetics laboratories, including:
1. DNA and RNA extraction from samples like blood and tissue.
2. Polymerase chain reaction (PCR) and its uses like detecting genetic mutations. Real-time PCR allows for quantitative analysis.
3. Techniques like ARMS-PCR, RFLP, and MLPA that can detect mutations, deletions, duplications to diagnose genetic disorders.
The techniques discussed have applications in prenatal diagnosis, forensic analysis like DNA fingerprinting, and identifying genetic causes of diseases. Molecular genetics labs aim to precisely analyze genetic information at the DNA and RNA level.
Types of PCR
There are several types of PCR that have been developed to address different needs:
1) Allele-specific PCR detects single nucleotide polymorphisms using primers designed to match one allele. 2) Multiplex PCR allows amplification of multiple targets simultaneously. 3) Nested PCR uses two sets of primers for two rounds of amplification to increase sensitivity. 4) Reverse transcription PCR amplifies DNA from RNA templates.
Types of PCR
There are several types of PCR that are used for different purposes:
1. Allele-specific PCR is used for single nucleotide polymorphism detection and DNA cloning. It requires prior knowledge of DNA sequence variations.
2. Multiplex PCR allows amplification of multiple DNA sequences simultaneously in a single reaction.
3. Nested PCR increases sensitivity by using two sets of primers in two sequential amplification reactions, with the second set binding internal to the first.
Some other types include reverse transcription PCR for amplifying RNA, touchdown PCR to reduce nonspecific amplification, and quantitative PCR for measuring starting DNA/RNA quantities.
New RNA tools for optimized CRISPR/Cas9 genome editing
The CRISPR/Cas9 system has emerged as one of the leading tools for modifying the genomes of organisms ranging from E. coli to humans. In this presentation, we discuss various methods for generating the crRNA and tracrRNA components that are required for guiding the Cas9 endonuclease to genomic targets. You will also learn how to optimize a new 2-part CRISPR RNA system from IDT that offers multiple benefits over other technologies.
PCR based Detection of Carbapenamases: Different primers and their utility
Carbapenems are Wide spectrum, β lactam class of antibiotic drug of last resort available as: imipenam, meropenam, doripenam and ertapenam. However, resistance to these drugs is emerging and may be mediated through: Efflux mechanism, Loss of porins, Change/ alterations in PBPs and specific β-lactamases - commonly known as carbapenamases.
Hoofdstuk 20 2008 deel 1
This chapter discusses DNA technology techniques including restriction enzymes, recombinant DNA, cloning genes, PCR, electrophoresis, and DNA sequencing. It provides an overview of using bacterial plasmids to clone genes, including restriction digestion to create sticky or blunt ends, ligation to insert DNA fragments into plasmids, transforming bacteria to reproduce the recombinant DNA, and selecting colonies containing the cloned gene. The chapter also describes applications of cloned genes and using restriction enzymes and ligation to create recombinant DNA.
Crop plants: DNA-free genome editing with CRISPR enzymes
This document summarizes a presentation on DNA-free genome editing with CRISPR enzymes. It discusses the evolution of genome editing techniques from random mutagenesis to targeted methods using ZFN, TALENs, and CRISPR/Cas9. The presentation focuses on a DNA-free approach using purified Cas9 protein and sgRNA to assemble CRISPR ribonucleoproteins (RNPs) for direct delivery into plant cells. It shows this method can induce targeted mutations in Arabidopsis, rice, and lettuce without leaving foreign DNA. RNPs generated mutations with high efficiency and specificity, and mutations were heritable after plant regeneration. Compared to other methods, DNA-free RNP delivery avoids issues like trans
Assembly and finishing
The document discusses genome assembly and finishing processes. It begins by outlining typical project goals of completely restoring the genome and producing a high-quality consensus sequence. It then describes the evolution of sequencing technologies from Sanger to newer platforms and their impact on draft assemblies. Key steps in the assembly and finishing process include library preparation, assembly, identifying gaps, and improving consensus quality.
Recombinant dna technology
This document discusses recombinant DNA technology and its applications. It covers key steps like cloning vectors, restriction modification systems, DNA ligases, transformation, and screening cDNA libraries to identify genes. Examples are given of recombinant proteins used as drugs to treat diseases like growth hormone deficiency, diabetes, and hemophilia. The last section briefly discusses genetic modification of animals and plants, and debates around genetically engineered salmon.
Transcription and translation
The document describes the process of transcription and translation. RNA polymerase in the nucleus reads DNA and produces mRNA, which exits into the cytoplasm. The ribosome then reads the mRNA and uses tRNA to assemble amino acids into a protein according to the mRNA's codon sequence.
RNA Folding
RNA folding during transcription can affect intrinsic transcription termination. The rate of RNA elongation influences the folding pathway, as the 5' end folds before the 3' end is synthesized. This can create kinetic traps that regulate biological processes like translation. RNA folding occurs inside the transcription elongation complex, and the constraints of this environment influence the folding of the termination hairpin. Weak RNA-RNA interactions competing with hairpin formation can also affect termination efficiency.
Rna Folding
RNA folding is affected by transcription elongation. As the RNA is synthesized within the elongation complex, its ability to fold is constrained. This can affect downstream processes like intrinsic transcription termination that depend on the formation of RNA hairpin structures. Experiments using RNase T1 to probe RNA structures in elongation complexes at different positions show that the hairpin involved in intrinsic termination begins to form early but may not fully fold until after the RNA is released from the elongation complex. Competing RNA-RNA interactions or kinetic constraints of hairpin folding within the elongation complex can influence the efficiency of intrinsic termination.
Polymerase chain reaction principles and practice
Polymerase chain reaction , its principles and its medical applications for medical and dental students
Pyrosequencing slide presentation rev3.
Pyrosequencing was evaluated as an alternative to Sanger sequencing for HIV drug resistance genotyping. Three assays were designed to detect mutations in the protease gene using a 356 bp amplicon and three sequencing primers. The assays demonstrated good linearity and sensitivity below 5% in quantifying mixed bases. However, read lengths were limited due to signal degradation, making it difficult to sequence large amplicons. Overall, pyrosequencing showed promise as a method for HIV genotyping but further optimization was needed to improve read lengths and mixed variant detection.
2011-Molecularmarker (1).ppt
This document discusses genetic markers and molecular markers used in plant breeding. It defines genetic markers as any phenotypic difference controlled by genes that can be used to study inheritance or select for traits. The two main types of genetic markers are morphological markers, which are visible traits, and molecular markers, which detect variations at the DNA or protein level. Several types of molecular markers are described in detail, including RFLP, RAPD, AFLP, SSR, and SNP markers. The document compares the characteristics of different marker types and outlines desirable properties for genetic markers, such as being polymorphic, co-dominant, and easy and cost-effective to detect.
2011-Molecularmarkerpppppppppppppppppt.ppt
This document discusses genetic markers and molecular markers used in plant breeding. It defines genetic markers as any phenotypic difference controlled by genes that can be used to study inheritance or select for traits. The two main types of genetic markers are morphological markers, which are visible traits, and molecular markers, which detect variations at the DNA or protein level. Several types of molecular markers are described in detail, including RFLP, RAPD, AFLP, SSR, and SNP markers. The document compares the characteristics of different marker types and outlines desirable properties for genetic markers, such as being polymorphic, co-dominant, and easy and cost-effective to detect.
rnaseq_from_babelomics
This document discusses RNA-Seq data analysis using Babelomics 5 software. It describes the typical RNA-Seq data analysis pipeline which includes sequence preprocessing, mapping, quantification, normalization, differential expression analysis, and functional profiling. It also discusses common file formats used like Fastq, BAM, and count matrices. Normalization methods like RPKM and TMM are explained. The document provides an overview of the RNA-Seq data analysis capabilities in Babelomics 5.
Similar to CRISPR.pptx (20)
Advenced molecular techniques in molecular medical genetics laboratory
Advenced molecular techniques in molecular medical genetics laboratory
New RNA tools for optimized CRISPR/Cas9 genome editing
New RNA tools for optimized CRISPR/Cas9 genome editing
PCR based Detection of Carbapenamases: Different primers and their utility
PCR based Detection of Carbapenamases: Different primers and their utility
Crop plants: DNA-free genome editing with CRISPR enzymes
Crop plants: DNA-free genome editing with CRISPR enzymes
Recently uploaded
在线办理(英国UEA毕业证书)东英格利亚大学毕业证成绩单一模一样
学校原件一模一样【微信：741003700 】《(英国UEA毕业证书)东英格利亚大学毕业证成绩单》【微信：741003700 】学位证，留信认证（真实可查，永久存档）原件一模一样纸张工艺/offer、雅思、外壳等材料/诚信可靠,可直接看成品样本，帮您解决无法毕业带来的各种难题！外壳，原版制作，诚信可靠，可直接看成品样本。行业标杆！精益求精，诚心合作，真诚制作！多年品质 ,按需精细制作，24小时接单,全套进口原装设备。十五年致力于帮助留学生解决难题，包您满意。
本公司拥有海外各大学样板无数，能完美还原。
1:1完美还原海外各大学毕业材料上的工艺：水印，阴影底纹，钢印LOGO烫金烫银，LOGO烫金烫银复合重叠。文字图案浮雕、激光镭射、紫外荧光、温感、复印防伪等防伪工艺。材料咨询办理、认证咨询办理请加学历顾问Q/微741003700
【主营项目】
一.毕业证【q微741003700】成绩单、使馆认证、教育部认证、雅思托福成绩单、学生卡等！
二.真实使馆公证(即留学回国人员证明,不成功不收费)
三.真实教育部学历学位认证（教育部存档！教育部留服网站永久可查）
四.办理各国各大学文凭(一对一专业服务,可全程监控跟踪进度)
如果您处于以下几种情况：
◇在校期间，因各种原因未能顺利毕业……拿不到官方毕业证【q/微741003700】
◇面对父母的压力，希望尽快拿到；
◇不清楚认证流程以及材料该如何准备；
◇回国时间很长，忘记办理；
◇回国马上就要找工作，办给用人单位看；
◇企事业单位必须要求办理的
◇需要报考公务员、购买免税车、落转户口
◇申请留学生创业基金
留信网认证的作用:
1:该专业认证可证明留学生真实身份
2:同时对留学生所学专业登记给予评定
3:国家专业人才认证中心颁发入库证书
4:这个认证书并且可以归档倒地方
5:凡事获得留信网入网的信息将会逐步更新到个人身份内，将在公安局网内查询个人身份证信息后，同步读取人才网入库信息
6:个人职称评审加20分
7:个人信誉贷款加10分
8:在国家人才网主办的国家网络招聘大会中纳入资料，供国家高端企业选择人才
Antyodaya saral portal haryana govt schemes
The Antyodaya Saral Haryana Portal is a pioneering initiative by the Government of Haryana aimed at providing citizens with seamless access to a wide range of government services
Bharat Mata - History of Indian culture.pdf
Bharat Mata Channel is an initiative towards keeping the culture of this country alive. Our effort is to spread the knowledge of Indian history, culture, religion and Vedas to the masses.
Milton Keynes Hospital Charity - A guide to leaving a gift in your Will
A forever thank you in your Will to Milton Keynes University Hospital can change lives for years to come.
Spending in the 340B Drug Pricing Program, 2010 to 2021
Presentation by Rebecca Sachs and Joshua Varcie, analysts in CBO’s Health Analysis Division, at the 13th Annual Conference of the American Society of Health Economists.
加急办理华威大学毕业证硕士文凭证书原版一模一样
原版一模一样【微信：741003700 】【华威大学毕业证硕士文凭证书】【微信：741003700 】学位证，留信认证（真实可查，永久存档）offer、雅思、外壳等材料/诚信可靠,可直接看成品样本，帮您解决无法毕业带来的各种难题！外壳，原版制作，诚信可靠，可直接看成品样本。行业标杆！精益求精，诚心合作，真诚制作！多年品质 ,按需精细制作，24小时接单,全套进口原装设备。十五年致力于帮助留学生解决难题，包您满意。
本公司拥有海外各大学样板无数，能完美还原海外各大学 Bachelor Diploma degree, Master Degree Diploma
1:1完美还原海外各大学毕业材料上的工艺：水印，阴影底纹，钢印LOGO烫金烫银，LOGO烫金烫银复合重叠。文字图案浮雕、激光镭射、紫外荧光、温感、复印防伪等防伪工艺。材料咨询办理、认证咨询办理请加学历顾问Q/微741003700
留信网认证的作用:
1:该专业认证可证明留学生真实身份
2:同时对留学生所学专业登记给予评定
3:国家专业人才认证中心颁发入库证书
4:这个认证书并且可以归档倒地方
5:凡事获得留信网入网的信息将会逐步更新到个人身份内，将在公安局网内查询个人身份证信息后，同步读取人才网入库信息
6:个人职称评审加20分
7:个人信誉贷款加10分
8:在国家人才网主办的国家网络招聘大会中纳入资料，供国家高端企业选择人才
一比一原版(theauckland毕业证书)新西兰奥克兰大学毕业证成绩单如何办理
原版定制【微信:741003700】《(theauckland毕业证书)新西兰奥克兰大学毕业证学位证成绩单》【微信:741003700】成绩单 、雅思、外壳、留信学历认证永久存档查询，采用学校原版纸张、特殊工艺完全按照原版一比一制作（包括：隐形水印，阴影底纹，钢印LOGO烫金烫银，LOGO烫金烫银复合重叠，文字图案浮雕，激光镭射，紫外荧光，温感，复印防伪）行业标杆！精益求精，诚心合作，真诚制作！多年品质 ,按需精细制作，24小时接单,全套进口原装设备，十五年致力于帮助留学生解决难题，业务范围有加拿大、英国、澳洲、韩国、美国、新加坡，新西兰等学历材料，包您满意。
【业务选择办理准则】
一、工作未确定，回国需先给父母、亲戚朋友看下文凭的情况，办理一份毕业证【Q微信741003700】文凭即可
二、回国进私企、外企、自己做生意的情况，这些单位是不查询毕业证真伪的，而且国内没有渠道去查询国外文凭的真假，也不需要提供真实教育部认证。鉴于此，办理一份毕业证【Q微信741003700】即可
三、进国企，银行，事业单位，考公务员等等，这些单位是必需要提供真实教育部认证的，办理教育部认证所需资料众多且烦琐，所有材料您都必须提供原件，我们凭借丰富的经验，快捷的绿色通道帮您快速整合材料，让您少走弯路。
留信网认证的作用:
1:该专业认证可证明留学生真实身份
2:同时对留学生所学专业登记给予评定
3:国家专业人才认证中心颁发入库证书
4:这个认证书并且可以归档倒地方
5:凡事获得留信网入网的信息将会逐步更新到个人身份内，将在公安局网内查询个人身份证信息后，同步读取人才网入库信息
6:个人职称评审加20分
7:个人信誉贷款加10分
8:在国家人才网主办的国家网络招聘大会中纳入资料，供国家高端企业选择人才
【关于价格问题（保证一手价格）】
我们所定的价格是非常合理的，而且我们现在做得单子大多数都是代理和回头客户介绍的所以一般现在有新的单子 我给客户的都是第一手的代理价格，因为我想坦诚对待大家 不想跟大家在价格方面浪费时间
对于老客户或者被老客户介绍过来的朋友，我们都会适当给一些优惠。
Contributi dei parlamentari del PD - Contributi L. 3/2019
DI SEGUITO SONO PUBBLICATI, AI SENSI DELL'ART. 11 DELLA LEGGE N. 3/2019, GLI IMPORTI RICEVUTI DALL'ENTRATA IN VIGORE DELLA SUDDETTA NORMA (31/01/2019) E FINO AL MESE SOLARE ANTECEDENTE QUELLO DELLA PUBBLICAZIONE SUL PRESENTE SITO
一比一原版(Adelaide毕业证)阿德莱德大学毕业证如何办理
原件一模一样【微信：95270640】【阿德莱德大学毕业证Adelaide学位证成绩单】【微信：95270640】（留信学历认证永久存档查询）采用学校原版纸张、特殊工艺完全按照原版一比一制作（包括：隐形水印，阴影底纹，钢印LOGO烫金烫银，LOGO烫金烫银复合重叠，文字图案浮雕，激光镭射，紫外荧光，温感，复印防伪）行业标杆！精益求精，诚心合作，真诚制作！多年品质 ,按需精细制作，24小时接单,全套进口原装设备，十五年致力于帮助留学生解决难题，业务范围有加拿大、英国、澳洲、韩国、美国、新加坡，新西兰等学历材料，包您满意。
【业务选择办理准则】
一、工作未确定，回国需先给父母、亲戚朋友看下文凭的情况，办理一份就读学校的毕业证【微信：95270640】文凭即可
二、回国进私企、外企、自己做生意的情况，这些单位是不查询毕业证真伪的，而且国内没有渠道去查询国外文凭的真假，也不需要提供真实教育部认证。鉴于此，办理一份毕业证【微信：95270640】即可
三、进国企，银行，事业单位，考公务员等等，这些单位是必需要提供真实教育部认证的，办理教育部认证所需资料众多且烦琐，所有材料您都必须提供原件，我们凭借丰富的经验，快捷的绿色通道帮您快速整合材料，让您少走弯路。
留信网认证的作用:
1:该专业认证可证明留学生真实身份【微信：95270640】
2:同时对留学生所学专业登记给予评定
3:国家专业人才认证中心颁发入库证书
4:这个认证书并且可以归档倒地方
5:凡事获得留信网入网的信息将会逐步更新到个人身份内，将在公安局网内查询个人身份证信息后，同步读取人才网入库信息
6:个人职称评审加20分
7:个人信誉贷款加10分
8:在国家人才网主办的国家网络招聘大会中纳入资料，供国家高端企业选择人才
→ 【关于价格问题（保证一手价格）
我们所定的价格是非常合理的，而且我们现在做得单子大多数都是代理和回头客户介绍的所以一般现在有新的单子 我给客户的都是第一手的代理价格，因为我想坦诚对待大家 不想跟大家在价格方面浪费时间
对于老客户或者被老客户介绍过来的朋友，我们都会适当给一些优惠。
选择实体注册公司办理，更放心，更安全！我们的承诺：可来公司面谈，可签订合同，会陪同客户一起到教育部认证窗口递交认证材料，客户在教育部官方认证查询网站查询到认证通过结果后付款，不成功不收费！
办理阿德莱德大学毕业证毕业证假文凭Adelaide学位证【微信：95270640 】外观非常精致，由特殊纸质材料制成，上面印有校徽、校名、毕业生姓名、专业等信息。
办理阿德莱德大学毕业证Adelaide学位证毕业证假文凭【微信：95270640 】格式相对统一，各专业都有相应的模板。通常包括以下部分：
校徽：象征着学校的荣誉和传承。
校名:学校英文全称
授予学位：本部分将注明获得的具体学位名称。
毕业生姓名：这是最重要的信息之一，标志着该证书是由特定人员获得的。
颁发日期：这是毕业正式生效的时间，也代表着毕业生学业的结束。
其他信息：根据不同的专业和学位，可能会有一些特定的信息或章节。
办理阿德莱德大学毕业证毕业证假文凭Adelaide学位证【微信：95270640 】价值很高，需要妥善保管。一般来说，应放置在安全、干燥、防潮的地方，避免长时间暴露在阳光下。如需使用，最好使用复印件而不是原件，以免丢失。
综上所述，办理阿德莱德大学毕业证毕业证假文凭Adelaide学位证【微信：95270640 】是证明身份和学历的高价值文件。外观简单庄重，格式统一，包括重要的个人信息和发布日期。对持有人来说，妥善保管是非常重要的。
2024: The FAR - Federal Acquisition Regulations, Part 41
Jennifer Schaus and Associates hosts a complimentary webinar series on The FAR in 2024. Join the webinars on Wednesdays and Fridays at noon, eastern.
Recordings are on YouTube and the company website.
https://www.youtube.com/@jenniferschaus/videos
原版制作(Hope毕业证书)利物浦霍普大学毕业证文凭证书一模一样
学校原件一模一样【微信：741003700 】《(Hope毕业证书)利物浦霍普大学毕业证文凭证书》【微信：741003700 】学位证，留信认证（真实可查，永久存档）原件一模一样纸张工艺/offer、雅思、外壳等材料/诚信可靠,可直接看成品样本，帮您解决无法毕业带来的各种难题！外壳，原版制作，诚信可靠，可直接看成品样本。行业标杆！精益求精，诚心合作，真诚制作！多年品质 ,按需精细制作，24小时接单,全套进口原装设备。十五年致力于帮助留学生解决难题，包您满意。
本公司拥有海外各大学样板无数，能完美还原。
1:1完美还原海外各大学毕业材料上的工艺：水印，阴影底纹，钢印LOGO烫金烫银，LOGO烫金烫银复合重叠。文字图案浮雕、激光镭射、紫外荧光、温感、复印防伪等防伪工艺。材料咨询办理、认证咨询办理请加学历顾问Q/微741003700
【主营项目】
一.毕业证【q微741003700】成绩单、使馆认证、教育部认证、雅思托福成绩单、学生卡等！
二.真实使馆公证(即留学回国人员证明,不成功不收费)
三.真实教育部学历学位认证（教育部存档！教育部留服网站永久可查）
四.办理各国各大学文凭(一对一专业服务,可全程监控跟踪进度)
如果您处于以下几种情况：
◇在校期间，因各种原因未能顺利毕业……拿不到官方毕业证【q/微741003700】
◇面对父母的压力，希望尽快拿到；
◇不清楚认证流程以及材料该如何准备；
◇回国时间很长，忘记办理；
◇回国马上就要找工作，办给用人单位看；
◇企事业单位必须要求办理的
◇需要报考公务员、购买免税车、落转户口
◇申请留学生创业基金
留信网认证的作用:
1:该专业认证可证明留学生真实身份
2:同时对留学生所学专业登记给予评定
3:国家专业人才认证中心颁发入库证书
4:这个认证书并且可以归档倒地方
5:凡事获得留信网入网的信息将会逐步更新到个人身份内，将在公安局网内查询个人身份证信息后，同步读取人才网入库信息
6:个人职称评审加20分
7:个人信誉贷款加10分
8:在国家人才网主办的国家网络招聘大会中纳入资料，供国家高端企业选择人才
在线办理(西班牙UPV毕业证书)瓦伦西亚理工大学毕业证毕业完成信一模一样
学校原件一模一样【微信：741003700 】《(西班牙UPV毕业证书)瓦伦西亚理工大学毕业证》【微信：741003700 】学位证，留信认证（真实可查，永久存档）原件一模一样纸张工艺/offer、雅思、外壳等材料/诚信可靠,可直接看成品样本，帮您解决无法毕业带来的各种难题！外壳，原版制作，诚信可靠，可直接看成品样本。行业标杆！精益求精，诚心合作，真诚制作！多年品质 ,按需精细制作，24小时接单,全套进口原装设备。十五年致力于帮助留学生解决难题，包您满意。
本公司拥有海外各大学样板无数，能完美还原。
1:1完美还原海外各大学毕业材料上的工艺：水印，阴影底纹，钢印LOGO烫金烫银，LOGO烫金烫银复合重叠。文字图案浮雕、激光镭射、紫外荧光、温感、复印防伪等防伪工艺。材料咨询办理、认证咨询办理请加学历顾问Q/微741003700
【主营项目】
一.毕业证【q微741003700】成绩单、使馆认证、教育部认证、雅思托福成绩单、学生卡等！
二.真实使馆公证(即留学回国人员证明,不成功不收费)
三.真实教育部学历学位认证（教育部存档！教育部留服网站永久可查）
四.办理各国各大学文凭(一对一专业服务,可全程监控跟踪进度)
如果您处于以下几种情况：
◇在校期间，因各种原因未能顺利毕业……拿不到官方毕业证【q/微741003700】
◇面对父母的压力，希望尽快拿到；
◇不清楚认证流程以及材料该如何准备；
◇回国时间很长，忘记办理；
◇回国马上就要找工作，办给用人单位看；
◇企事业单位必须要求办理的
◇需要报考公务员、购买免税车、落转户口
◇申请留学生创业基金
留信网认证的作用:
1:该专业认证可证明留学生真实身份
2:同时对留学生所学专业登记给予评定
3:国家专业人才认证中心颁发入库证书
4:这个认证书并且可以归档倒地方
5:凡事获得留信网入网的信息将会逐步更新到个人身份内，将在公安局网内查询个人身份证信息后，同步读取人才网入库信息
6:个人职称评审加20分
7:个人信誉贷款加10分
8:在国家人才网主办的国家网络招聘大会中纳入资料，供国家高端企业选择人才
IEA World Energy Investment June 2024- Statistics
IEA World Energy Investment Report 2024
Publication: June 2024
2024: The FAR - Federal Acquisition Regulations, Part 42
Jennifer Schaus and Associates hosts a complimentary webinar series on The FAR in 2024. Join the webinars on Wednesdays and Fridays at noon, eastern.
Recordings are on YouTube and the company website.
https://www.youtube.com/@jenniferschaus/videos
快速办理(Bristol毕业证书)布里斯托大学毕业证Offer一模一样
学校原件一模一样【微信：741003700 】《(Bristol毕业证书)布里斯托大学毕业证》【微信：741003700 】学位证，留信认证（真实可查，永久存档）原件一模一样纸张工艺/offer、雅思、外壳等材料/诚信可靠,可直接看成品样本，帮您解决无法毕业带来的各种难题！外壳，原版制作，诚信可靠，可直接看成品样本。行业标杆！精益求精，诚心合作，真诚制作！多年品质 ,按需精细制作，24小时接单,全套进口原装设备。十五年致力于帮助留学生解决难题，包您满意。
本公司拥有海外各大学样板无数，能完美还原。
1:1完美还原海外各大学毕业材料上的工艺：水印，阴影底纹，钢印LOGO烫金烫银，LOGO烫金烫银复合重叠。文字图案浮雕、激光镭射、紫外荧光、温感、复印防伪等防伪工艺。材料咨询办理、认证咨询办理请加学历顾问Q/微741003700
【主营项目】
一.毕业证【q微741003700】成绩单、使馆认证、教育部认证、雅思托福成绩单、学生卡等！
二.真实使馆公证(即留学回国人员证明,不成功不收费)
三.真实教育部学历学位认证（教育部存档！教育部留服网站永久可查）
四.办理各国各大学文凭(一对一专业服务,可全程监控跟踪进度)
如果您处于以下几种情况：
◇在校期间，因各种原因未能顺利毕业……拿不到官方毕业证【q/微741003700】
◇面对父母的压力，希望尽快拿到；
◇不清楚认证流程以及材料该如何准备；
◇回国时间很长，忘记办理；
◇回国马上就要找工作，办给用人单位看；
◇企事业单位必须要求办理的
◇需要报考公务员、购买免税车、落转户口
◇申请留学生创业基金
留信网认证的作用:
1:该专业认证可证明留学生真实身份
2:同时对留学生所学专业登记给予评定
3:国家专业人才认证中心颁发入库证书
4:这个认证书并且可以归档倒地方
5:凡事获得留信网入网的信息将会逐步更新到个人身份内，将在公安局网内查询个人身份证信息后，同步读取人才网入库信息
6:个人职称评审加20分
7:个人信誉贷款加10分
8:在国家人才网主办的国家网络招聘大会中纳入资料，供国家高端企业选择人才
2024: The FAR - Federal Acquisition Regulations, Part 40
Jennifer Schaus and Associates hosts a complimentary webinar series on The FAR in 2024. Join the webinars on Wednesdays and Fridays at noon, eastern.
Recordings are on YouTube and the company website.
https://www.youtube.com/@jenniferschaus/videos
CBO's Immigration Projections - Presentation
Presentation by Julie Topoleski, CBO’s Director of Labor, Income Security, and Long-Term Analysis, at the 16th Annual Meeting of the OECD Working Party of Parliamentary Budget Officials and Independent Fiscal Institutions.
一比一原版英国阿伯丁大学毕业证(AU毕业证书)学历如何办理
退学办理文凭【微信号:176555708】【(AU毕业证书)】【微信号:176555708】《成绩单、外壳、offer、真实留信官方学历认证（永久存档/真实可查）》采用学校原版纸张、特殊工艺完全按照原版一比一制作（包括：隐形水印，阴影底纹，钢印LOGO烫金烫银，LOGO烫金烫银复合重叠，文字图案浮雕，激光镭射，紫外荧光，温感，复印防伪）行业标杆！精益求精，诚心合作，真诚制作！多年品质 ,按需精细制作，24小时接单,全套进口原装设备，十五年致力于帮助留学生解决难题，业务范围有加拿大、英国、澳洲、韩国、美国、新加坡，新西兰等学历材料，包您满意。
【我们承诺采用的是学校原版纸张（纸质、底色、纹路）我们拥有全套进口原装设备，特殊工艺都是采用不同机器制作，仿真度基本可以达到100%，所有工艺效果都可提前给客户展示，不满意可以根据客户要求进行调整，直到满意为止！】
【业务选择办理准则】
一、工作未确定，回国需先给父母、亲戚朋友看下文凭的情况，办理一份就读学校的毕业证【微信号:176555708】文凭即可
二、回国进私企、外企、自己做生意的情况，这些单位是不查询毕业证真伪的，而且国内没有渠道去查询国外文凭的真假，也不需要提供真实教育部认证。鉴于此，办理一份毕业证【微信号:176555708】即可
三、进国企，银行，事业单位，考公务员等等，这些单位是必需要提供真实教育部认证的，办理教育部认证所需资料众多且烦琐，所有材料您都必须提供原件，我们凭借丰富的经验，快捷的绿色通道帮您快速整合材料，让您少走弯路。
留信网认证的作用:
1:该专业认证可证明留学生真实身份
2:同时对留学生所学专业登记给予评定
3:国家专业人才认证中心颁发入库证书
4:这个认证书并且可以归档倒地方
5:凡事获得留信网入网的信息将会逐步更新到个人身份内，将在公安局网内查询个人身份证信息后，同步读取人才网入库信息
6:个人职称评审加20分
7:个人信誉贷款加10分
8:在国家人才网主办的国家网络招聘大会中纳入资料，供国家高端企业选择人才
留信网服务项目：
1、留学生专业人才库服务（留信分析）
2、国（境）学习人员提供就业推荐信服务
3、留学人员区块链存储服务
【关于价格问题（保证一手价格）】
我们所定的价格是非常合理的，而且我们现在做得单子大多数都是代理和回头客户介绍的所以一般现在有新的单子 我给客户的都是第一手的代理价格，因为我想坦诚对待大家 不想跟大家在价格方面浪费时间
对于老客户或者被老客户介绍过来的朋友，我们都会适当给一些优惠。
选择实体注册公司办理，更放心，更安全！我们的承诺：客户在留信官方认证查询网站查询到认证通过结果后付款，不成功不收费！
Recently uploaded (20)
Milton Keynes Hospital Charity - A guide to leaving a gift in your Will
Milton Keynes Hospital Charity - A guide to leaving a gift in your Will
Spending in the 340B Drug Pricing Program, 2010 to 2021
Spending in the 340B Drug Pricing Program, 2010 to 2021
Contributi dei parlamentari del PD - Contributi L. 3/2019
Contributi dei parlamentari del PD - Contributi L. 3/2019
2024: The FAR - Federal Acquisition Regulations, Part 41
2024: The FAR - Federal Acquisition Regulations, Part 41
2024: The FAR - Federal Acquisition Regulations, Part 42
2024: The FAR - Federal Acquisition Regulations, Part 42
2024: The FAR - Federal Acquisition Regulations, Part 40
2024: The FAR - Federal Acquisition Regulations, Part 40
CRISPR.pptx
- 1. CRISPR
- 2. Requirements 1. Primer sets for gRNA amplicon, vector backbone and colony PCR for recombinant vector with gRNA 2. Hi-Fidelity long range polymerases like Q5/ Phusion 3. Gibson Assembler with master mix having, T5 exonuclease, taq ligase and Phusion polymerase
- 3. Primer sets 1)Primers for gRNA amplicon dLarp7 gRNA8.0 FP atatccgggtgaacttcgGTCAGGAGCCCGCGACATCAGgttttagagctagaaatag dLarp7 gRNA6.4 RP tctagctctaaaacCCATGGACTGAGTCACTTTCcgacgttaaattgaaaatagg 2)Primers for backbone amplicon dLarp7 GA FP cctattttcaatttaacgtcggggtcttcgagaagacctgttttagagctagaaatag dLarp7 GA RP ctatttctagctctaaaacaggtcttctcgaagaccccgaagttcacccggatat 3)Colony PCR for recombinant vector with gRNA Test dLarp7 gRNA 8.0 FP CAGGAGCCCGCGACATCAG Test dLarp7 gRNA 6.4 RP CCATGGACTGAGTCACTTTC
- 4. Method • Generate recombinant pCDF4 with 2 tandem gRNAs against dLarp7 • At the facility - Microinject it into BL 25709/25710 with attP docking site for phiC31 integrase- mediated transformation on 2/3rd chromosome • gRNA flies x Act5c Cas9 flies on X chromosome (Need suggestion to which marker should be present in the Cas9 stock) to obtain F1 progeny with del • F1 x double bal stock yield F2 which will be used for screening the deletion through PCR