Optimising parameters associated with a high through put FRET assay for identifying Tryp REL 1 antagonists, viz.
1. Ligation conditions, cf. buffer composition e.g. pH; ligation time etc
2. Mixing and matching fluorophores to maximise S:N and hence assay sensitivity
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.
Applications of pcr in detection of food borne pathogens and gm foodsMozhi Arasu
This document provides an overview of polymerase chain reaction (PCR) methods for detecting pathogens in food. It discusses sample preparation, primer design, factors that affect PCR, and types of PCR methods. Specific examples are given of pathogens detected by PCR in various foods like dairy, meat, seafood, grains and vegetables. Methods for visualizing and analyzing PCR results are also summarized. The document concludes with a comparison of PCR to conventional microbiology methods and a discussion of validation, potential issues, and setting up a PCR laboratory.
The document discusses the Polymerase Chain Reaction (PCR) technique. It was invented by Kary Mullis in 1985. PCR allows for targeted amplification of specific DNA sequences. It works by using DNA polymerase to make copies of a targeted region of DNA defined by primer sequences. Through repeated heating and cooling cycles, millions of copies of the target DNA can be produced, allowing it to be analyzed. Taq polymerase, an enzyme from thermophilic bacteria, is used as it is heat-stable and allows the reaction to take place.
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.
The document discusses efficient chemical synthesis of long and modified RNA oligonucleotides using TC-RNA chemistry. It describes how TC-RNA chemistry allows for robust and easy RNA synthesis similar to DNA synthesis, with high efficiency and quality RNA that can incorporate modifications. It provides examples of synthesizing various RNA oligos up to 113 nucleotides long for applications like ribozymes and CRISPR-Cas9 and shows their activity matches in vitro transcribed RNA. Mass spectrometry is used to analyze purity and molecular weight matching calculations.
Polymerase Chain Reaction (PCR) is a technique used to amplify small amounts of DNA sequences. It involves repeated cycles of heating and cooling of the DNA sample to denature and replicate the target DNA. Each cycle doubles the amount of target DNA, exponentially increasing its quantity for analysis. PCR uses primers, DNA polymerase, and dNTPs to selectively amplify the target DNA sequence. It has revolutionized molecular biology and is widely used for DNA cloning, detection of genetic diseases and mutations, forensic analysis, and more.
Optimising parameters associated with a high through put FRET assay for identifying Tryp REL 1 antagonists, viz.
1. Ligation conditions, cf. buffer composition e.g. pH; ligation time etc
2. Mixing and matching fluorophores to maximise S:N and hence assay sensitivity
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.
Applications of pcr in detection of food borne pathogens and gm foodsMozhi Arasu
This document provides an overview of polymerase chain reaction (PCR) methods for detecting pathogens in food. It discusses sample preparation, primer design, factors that affect PCR, and types of PCR methods. Specific examples are given of pathogens detected by PCR in various foods like dairy, meat, seafood, grains and vegetables. Methods for visualizing and analyzing PCR results are also summarized. The document concludes with a comparison of PCR to conventional microbiology methods and a discussion of validation, potential issues, and setting up a PCR laboratory.
The document discusses the Polymerase Chain Reaction (PCR) technique. It was invented by Kary Mullis in 1985. PCR allows for targeted amplification of specific DNA sequences. It works by using DNA polymerase to make copies of a targeted region of DNA defined by primer sequences. Through repeated heating and cooling cycles, millions of copies of the target DNA can be produced, allowing it to be analyzed. Taq polymerase, an enzyme from thermophilic bacteria, is used as it is heat-stable and allows the reaction to take place.
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.
The document discusses efficient chemical synthesis of long and modified RNA oligonucleotides using TC-RNA chemistry. It describes how TC-RNA chemistry allows for robust and easy RNA synthesis similar to DNA synthesis, with high efficiency and quality RNA that can incorporate modifications. It provides examples of synthesizing various RNA oligos up to 113 nucleotides long for applications like ribozymes and CRISPR-Cas9 and shows their activity matches in vitro transcribed RNA. Mass spectrometry is used to analyze purity and molecular weight matching calculations.
Polymerase Chain Reaction (PCR) is a technique used to amplify small amounts of DNA sequences. It involves repeated cycles of heating and cooling of the DNA sample to denature and replicate the target DNA. Each cycle doubles the amount of target DNA, exponentially increasing its quantity for analysis. PCR uses primers, DNA polymerase, and dNTPs to selectively amplify the target DNA sequence. It has revolutionized molecular biology and is widely used for DNA cloning, detection of genetic diseases and mutations, forensic analysis, and more.
Real time pcr, reverse transcripta pcr, gene expression methods and microarrayharrisonjoshua
Real-time PCR, reverse transcriptase PCR, gene expression methods and microarrays. The document discusses various methods for studying gene expression including real-time PCR, reverse transcriptase PCR, microarrays, and other related topics. Real-time PCR allows for quantification of gene expression in real-time and has advantages like a wide dynamic range and little inter-assay variation. Reverse transcriptase PCR is used to detect RNA expression by first converting RNA to cDNA then amplifying it with PCR. Microarrays allow studying the activity patterns of thousands of genes simultaneously on a DNA chip.
PCR (polymerase chain reaction) is a technique used to amplify a specific region of DNA. It involves repeated cycles of heating and cooling of the DNA sample to denature the DNA strands, allow primers to anneal to the target region, and extend new strands using a DNA polymerase. Each cycle doubles the number of copies of the target region, allowing millions of copies to be produced from a single DNA molecule. PCR was invented in 1985 and has many applications, including detecting DNA sequences and quantifying gene expression levels.
Polymerase chain reaction (PCR) is a technique used to amplify a specific region of DNA. It involves repeated cycles of heating and cooling of the DNA sample in the presence of primers and a DNA polymerase. Key developments included the discovery of thermostable DNA polymerases and commercialization of the PCR technique. PCR has widespread applications in research, forensics, disease diagnosis, and DNA sequencing due to its ability to selectively amplify DNA sequences.
PCR is a technique used to amplify a specific DNA sequence. It involves repeated cycles of heating and cooling of the DNA sample to separate and copy the DNA strands. Key components of PCR include primers, DNA polymerase, and dNTPs. Variations of PCR allow for applications such as detecting gene expression, sequencing DNA, and quantifying DNA. Limitations include errors during amplification and potential contamination issues.
The polymerase chain reaction (PCR) is a technique used to amplify a single copy of a DNA segment across several orders of magnitude. It was developed by Kary Mullis in 1983 and involves thermal cycling to separate DNA strands and allow a DNA polymerase to replicate the strands. This results in exponential amplification of the target DNA segment. PCR is now widely used in medical research and forensic sciences to amplify specific DNA regions.
The document provides instructions for using Gateway cloning technology to construct entry clones and expression clones for protein expression. Key steps include:
1. Constructing entry clones containing the gene of interest using restriction digestion/ligation or BP recombination.
2. Choosing a destination vector for protein expression in E. coli, mammalian cells, or baculovirus based on desired tags and expression system.
3. Transferring the gene of interest from the entry clone to the destination vector using LR recombination to generate an expression clone for protein production.
The document discusses the Gateway cloning system, which allows efficient transfer of genes between vectors using site-specific recombination. It describes how the system uses bacteriophage lambda integrase to catalyze recombination between att sites. Genes can be shuttled between an entry clone containing attL sites and a destination vector containing attR sites via an LR reaction. The Gateway system provides a simple and efficient way to clone genes into multiple expression vectors without restriction digestion or ligation.
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.
the speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionized molecular biology and made PCR the most useful and powerful technique with great spectrum of research and diagnostic applications.
Enzymes In Transcription, Translation and ReplicationAngsumanDas2
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.
The document discusses several topics related to DNA and genetics including DNA replication, restriction enzymes, gel electrophoresis, PCR, DNA sequencing, cloning, and gene expression. It provides figures to illustrate key concepts and techniques such as how restriction enzymes cut DNA, how gel electrophoresis separates DNA fragments by size, how PCR amplifies a target DNA sequence, and the process of cloning a gene into a bacterial plasmid.
Ultra stable lipid system for the study of P450 EnzymesEmek Blair, Ph.D.
A structured lipid system surrounding the enzymes which is stable at high temperature and acidic conditions is used to characterize P450 CYP119. These mimic the natural lipid bilayers that the enzymes naturally are stable in.
Lab talk 020410 inducing rel 1 to set up ht fret assay_progressLaurence Dawkins-Hall
The document discusses preliminary experiments to induce soluble rREL1 protein for use in high-throughput screening of small molecule compounds. It describes investigating the potency of 6 established lead molecules on cell viability and generating dose response data for Mordant Black 25. Conditions for expressing and purifying rREL1 under different lysis methods are explored. Plans are outlined for establishing a fluorescence-based ligase assay using labeled RNA fragments and optimizing various assay parameters.
The document discusses several topics related to DNA replication, genetic engineering, and gene regulation:
1) It describes the basic process of DNA replication, including the roles of helicase, primase, topoisomerase, and other proteins.
2) It explains some techniques used in genetic engineering, such as restriction enzymes cutting DNA at specific sites, ligation to join DNA fragments, and gel electrophoresis to separate DNA fragments by size.
3) It discusses mechanisms of gene regulation, including repression and activation of operons in response to metabolites and how this controls enzyme production for metabolic pathways like tryptophan and lactose synthesis.
1. more work at optimising expression and purification of rREL 1 for incorporation into a high throughput FRET assay for compound library screens to identify REL 1 antagonists
2. Regression analysis of titrants of compounds identified as demonstrating REL 1 inhibition by radiomimetic assay; quantification of efficacy by means of IC50
1. The document describes the core analysis steps for ChIP-seq and RNA-seq experiments, including trimming, alignment, peak calling, and downstream analyses like viewing data in a genome browser and identifying motifs.
2. It explains key ChIP-seq steps like sonication, immunoprecipitation of DNA-bound proteins, and use of control samples to identify true enrichment peaks.
3. It also outlines RNA-seq workflow involving poly-A selection, cDNA synthesis, and analysis of gene and transcript expression.
RNA editing as a drug target development of a high through put fluorescent as...Laurence Dawkins-Hall
This document describes the development and optimization of a fluorescence resonance energy transfer (FRET)-based assay to detect and quantify the ligation activity of RNA ligase 1 (REL1). The assay uses donor- and acceptor-labeled RNA oligos that are annealed to an RNA bridge to bring the fluorophores into close proximity, allowing FRET. Ligation by REL1 generates a contiguous dual-labeled species, while inhibition prevents ligation and abrogates FRET. The assay was statistically validated, and conditions such as pH, denaturant, and competitor oligo concentration were optimized to ensure specific and efficient ligation detection. Purified REL1-A2 protein fractions demonstrated significant ligation activity in the assay.
NYSAS Seminar LC-IR To Characterize Polymeric Excipients In Pharmaceutical F...mzhou45
This document describes an LC-IR technique for characterizing polymeric excipients in pharmaceutical formulations. The LC-IR system combines liquid chromatography separation with online infrared spectroscopy detection. It is used to characterize copolymer compositions, detect excipient degradation from hot melt extrusion processes, and study the stability of excipients like PEG. The LC-IR technique provides compositional information and identifies degradation products with molecular weight distributions. This allows understanding of excipient properties, degradation mechanisms, and process effects to ensure quality and stability of pharmaceutical formulations.
Real time pcr, reverse transcripta pcr, gene expression methods and microarrayharrisonjoshua
Real-time PCR, reverse transcriptase PCR, gene expression methods and microarrays. The document discusses various methods for studying gene expression including real-time PCR, reverse transcriptase PCR, microarrays, and other related topics. Real-time PCR allows for quantification of gene expression in real-time and has advantages like a wide dynamic range and little inter-assay variation. Reverse transcriptase PCR is used to detect RNA expression by first converting RNA to cDNA then amplifying it with PCR. Microarrays allow studying the activity patterns of thousands of genes simultaneously on a DNA chip.
PCR (polymerase chain reaction) is a technique used to amplify a specific region of DNA. It involves repeated cycles of heating and cooling of the DNA sample to denature the DNA strands, allow primers to anneal to the target region, and extend new strands using a DNA polymerase. Each cycle doubles the number of copies of the target region, allowing millions of copies to be produced from a single DNA molecule. PCR was invented in 1985 and has many applications, including detecting DNA sequences and quantifying gene expression levels.
Polymerase chain reaction (PCR) is a technique used to amplify a specific region of DNA. It involves repeated cycles of heating and cooling of the DNA sample in the presence of primers and a DNA polymerase. Key developments included the discovery of thermostable DNA polymerases and commercialization of the PCR technique. PCR has widespread applications in research, forensics, disease diagnosis, and DNA sequencing due to its ability to selectively amplify DNA sequences.
PCR is a technique used to amplify a specific DNA sequence. It involves repeated cycles of heating and cooling of the DNA sample to separate and copy the DNA strands. Key components of PCR include primers, DNA polymerase, and dNTPs. Variations of PCR allow for applications such as detecting gene expression, sequencing DNA, and quantifying DNA. Limitations include errors during amplification and potential contamination issues.
The polymerase chain reaction (PCR) is a technique used to amplify a single copy of a DNA segment across several orders of magnitude. It was developed by Kary Mullis in 1983 and involves thermal cycling to separate DNA strands and allow a DNA polymerase to replicate the strands. This results in exponential amplification of the target DNA segment. PCR is now widely used in medical research and forensic sciences to amplify specific DNA regions.
The document provides instructions for using Gateway cloning technology to construct entry clones and expression clones for protein expression. Key steps include:
1. Constructing entry clones containing the gene of interest using restriction digestion/ligation or BP recombination.
2. Choosing a destination vector for protein expression in E. coli, mammalian cells, or baculovirus based on desired tags and expression system.
3. Transferring the gene of interest from the entry clone to the destination vector using LR recombination to generate an expression clone for protein production.
The document discusses the Gateway cloning system, which allows efficient transfer of genes between vectors using site-specific recombination. It describes how the system uses bacteriophage lambda integrase to catalyze recombination between att sites. Genes can be shuttled between an entry clone containing attL sites and a destination vector containing attR sites via an LR reaction. The Gateway system provides a simple and efficient way to clone genes into multiple expression vectors without restriction digestion or ligation.
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.
the speed and ease of use, sensitivity, specificity and robustness of PCR has revolutionized molecular biology and made PCR the most useful and powerful technique with great spectrum of research and diagnostic applications.
Enzymes In Transcription, Translation and ReplicationAngsumanDas2
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.
The document discusses several topics related to DNA and genetics including DNA replication, restriction enzymes, gel electrophoresis, PCR, DNA sequencing, cloning, and gene expression. It provides figures to illustrate key concepts and techniques such as how restriction enzymes cut DNA, how gel electrophoresis separates DNA fragments by size, how PCR amplifies a target DNA sequence, and the process of cloning a gene into a bacterial plasmid.
Ultra stable lipid system for the study of P450 EnzymesEmek Blair, Ph.D.
A structured lipid system surrounding the enzymes which is stable at high temperature and acidic conditions is used to characterize P450 CYP119. These mimic the natural lipid bilayers that the enzymes naturally are stable in.
Lab talk 020410 inducing rel 1 to set up ht fret assay_progressLaurence Dawkins-Hall
The document discusses preliminary experiments to induce soluble rREL1 protein for use in high-throughput screening of small molecule compounds. It describes investigating the potency of 6 established lead molecules on cell viability and generating dose response data for Mordant Black 25. Conditions for expressing and purifying rREL1 under different lysis methods are explored. Plans are outlined for establishing a fluorescence-based ligase assay using labeled RNA fragments and optimizing various assay parameters.
The document discusses several topics related to DNA replication, genetic engineering, and gene regulation:
1) It describes the basic process of DNA replication, including the roles of helicase, primase, topoisomerase, and other proteins.
2) It explains some techniques used in genetic engineering, such as restriction enzymes cutting DNA at specific sites, ligation to join DNA fragments, and gel electrophoresis to separate DNA fragments by size.
3) It discusses mechanisms of gene regulation, including repression and activation of operons in response to metabolites and how this controls enzyme production for metabolic pathways like tryptophan and lactose synthesis.
1. more work at optimising expression and purification of rREL 1 for incorporation into a high throughput FRET assay for compound library screens to identify REL 1 antagonists
2. Regression analysis of titrants of compounds identified as demonstrating REL 1 inhibition by radiomimetic assay; quantification of efficacy by means of IC50
1. The document describes the core analysis steps for ChIP-seq and RNA-seq experiments, including trimming, alignment, peak calling, and downstream analyses like viewing data in a genome browser and identifying motifs.
2. It explains key ChIP-seq steps like sonication, immunoprecipitation of DNA-bound proteins, and use of control samples to identify true enrichment peaks.
3. It also outlines RNA-seq workflow involving poly-A selection, cDNA synthesis, and analysis of gene and transcript expression.
RNA editing as a drug target development of a high through put fluorescent as...Laurence Dawkins-Hall
This document describes the development and optimization of a fluorescence resonance energy transfer (FRET)-based assay to detect and quantify the ligation activity of RNA ligase 1 (REL1). The assay uses donor- and acceptor-labeled RNA oligos that are annealed to an RNA bridge to bring the fluorophores into close proximity, allowing FRET. Ligation by REL1 generates a contiguous dual-labeled species, while inhibition prevents ligation and abrogates FRET. The assay was statistically validated, and conditions such as pH, denaturant, and competitor oligo concentration were optimized to ensure specific and efficient ligation detection. Purified REL1-A2 protein fractions demonstrated significant ligation activity in the assay.
NYSAS Seminar LC-IR To Characterize Polymeric Excipients In Pharmaceutical F...mzhou45
This document describes an LC-IR technique for characterizing polymeric excipients in pharmaceutical formulations. The LC-IR system combines liquid chromatography separation with online infrared spectroscopy detection. It is used to characterize copolymer compositions, detect excipient degradation from hot melt extrusion processes, and study the stability of excipients like PEG. The LC-IR technique provides compositional information and identifies degradation products with molecular weight distributions. This allows understanding of excipient properties, degradation mechanisms, and process effects to ensure quality and stability of pharmaceutical formulations.
This document discusses cascade, ratio, and feedforward control techniques. Cascade control reduces the effect of specific disturbances by using a faster slave loop. Ratio control reduces the effect of feed flow rate changes. Feedforward control compensates for measured disturbances before they affect the process. The document provides examples of how each control technique can be applied and their advantages and limitations.
This document discusses cascade, ratio, and feedforward control techniques. Cascade control reduces the effect of specific disturbances by using a faster slave loop. Ratio control reduces the effect of feed flow rate changes. Feedforward control compensates for measured disturbances before they affect the process. The document provides examples of how each control technique can be applied and their advantages and limitations.
1. The document discusses mechanisms of substitution and elimination reactions for alkyl halides, including SN1, SN2, E1 and E2.
2. Key factors that determine the reaction mechanism are the structure of the alkyl halide (primary, secondary, tertiary), the nucleophilicity and basicity of the reaction conditions, and temperature.
3. SN1 proceeds through a carbocation intermediate and gives racemic products. SN2 is stereospecific. E1 and E2 are elimination reactions favored by strong bases and high temperatures.
1. The document discusses mechanisms of substitution and elimination reactions for alkyl halides, including SN1, SN2, E1 and E2.
2. Key factors that determine the reaction mechanism are the structure of the alkyl halide (primary, secondary, tertiary), the nucleophilicity and basicity of the reaction conditions, and temperature.
3. SN1 proceeds through a carbocation intermediate and gives racemic products. SN2 is stereospecific. E1 and E2 are elimination reactions that compete with substitution.
FermaseCALBTM10L is a recombinant Candida antarctica lipase B enzyme expressed in yeast cells that is supplied as a formulated enzyme concentrate. It has a minimum activity of 10,000 TBU/mL for tributyrin hydrolysis and 4,500 PLU/mL for propyllaurate synthesis. It exhibits good stereoselectivity for resolving chiral alcohols and amines and synthesizing fatty acid esters. It has high pH, temperature, and storage stability and is safe to handle.
The document describes the development of two TaqMan digital PCR liquid biopsy assays to detect mutations in the TERT promoter region associated with cancer. The assays target the C228T and C250T mutations. Testing showed the assays could reliably detect the mutations at levels as low as 0.1% mutation frequency in genomic DNA samples. Assay design was challenging due to the high GC content and repetitive sequences in the TERT promoter region. Optimization of thermal cycling conditions was needed for accurate detection of the mutations by digital PCR.
This document summarizes membrane separation technology used to purify anserine-carnosine extracted from chicken meat. It describes the background of membrane separation processes, materials used to prepare a chicken extract, various membrane types tested for separation, and results of experiments investigating the effect of operating pressure and flow rate on permeate flux and rejection rates of key components. It proposes a mathematical model to simulate experimental results and presents a process design for purifying anserine-carnosine from 5 tons of chicken carcass per day using membrane filtration.
Similar to Labtalk 101210 optimising fret assay_den_optimising r_rel 1 production_heat shock (20)
An evaluation of methods used to sequence pGEM template within core facilitie...Laurence Dawkins-Hall
A horizontal study by ABRF designed to investigate & collate cycle sequencing methods for the pGEM template and subsequent data output on automated sequencing platforms (cf. AB 3700 & AB 3100)
06 isafg p77 genome wide expression consequences of a disease resistance qtl ...Laurence Dawkins-Hall
Genome wide expression consequences of a disease resistance QTL are strongly influenced by the genetic background. The document discusses finding QTL genes in mice, with few genes having a large effect and many having small effects. It also discusses mouse models of trypanosomiasis and survival differences between parental strains and an F6 generation. Trypanosoma infection response loci were mapped between C57BL/6 and AJ and C57BL/6 and BALB/c mouse strains. A microarray study looked at gene expression differences between resistant C57BL/6 and susceptible AJ mice. The Daxx gene was found to be located within a Trypanosoma infection response QTL and had a polymorphism in its p53
Elucidating changes in gene expression in Tryp susceptible and resistant cattle during progression of tryp infection using Affymetrix gene expression Micro arrays
Genomic gene expression changes resulting from Trypanosomiasis: a horizontal study Examining expression changes elucidated by micro arrays in seminal tissues associated with the pathophysiology of Trypanosomiasis during disease progression
Systemic analysis of data combined from genetic qtl's and gene expression dat...Laurence Dawkins-Hall
Elucidating changes in gene expression by Micro array genomic sweeps of genetic QTLs linked to Tryp resistance in WT cattle to identify putative candidates underpinning pathophysiology
A systematic, data driven approach to the combined analysis of microarray and...Laurence Dawkins-Hall
The use of gene expression data from Micro arrays coupled with WT QTL's linked to Tryp resistance phenotypes in Cattle to elucidate pertinent genetic changes underpinning phenotype in putative candidate genes
Lab talk 201109 radioligand assay for validating in slico predicted rel1 anta...Laurence Dawkins-Hall
This document discusses identifying inhibitors of REL1, an essential enzyme in Trypanosoma brucei, the parasite that causes African sleeping sickness. It outlines objectives to screen compounds for REL1 inhibition and determine IC50 values. Several sets of compounds from various sources were tested, and some inhibitors with IC50 values in the low micromolar range were identified. Future work includes completing additional titrations and re-testing some previous assays to further characterize inhibitory compounds.
Lab talk 060809 radioligand assay to validate in slico predicted rel inhibito...Laurence Dawkins-Hall
The document outlines steps to identify inhibitors of REL1 through radioactive labelling assays. Several potential inhibitor sets were identified, including two batches of NCI compounds. Batch 1 compounds #117079 and #125908 showed variable but substantial inhibition at 10uM, and over 99% and 95% inhibition respectively at 100uM. Batch 2 compounds #45609, #162535 and #1698 showed over 95%, 95% and 80% inhibition at 10uM respectively. Further work included completing assays, screening additional concentrations, determining IC50 values for top inhibitors, and evaluating other inhibitor sets and growth effects.
Lab talk 190210 efficacy studies on radioligand hits_beginnings of fret assay...Laurence Dawkins-Hall
1. Titration of REL 1 antagonist hits identified by radio mimetic assay to quantify efficacy (IC50)
2. Exposition of HT FRET assay principles for large scale compound library screens to empirically identify REL 1 antagonist hits
Lab talk 020710 comparing bac r_rel 1 with e coli rrel 1 for use in fret assayLaurence Dawkins-Hall
Comparing activity of baculovirus and E Coli expressed rREL 1 fractions in context of HT FRET assay for screening compound libraries to identify REL1 antagonist hits
Rna editing as a drug target identification of inhibitors of rel 1 bsp 2010Laurence Dawkins-Hall
(1) RNA editing is essential in trypanosomatids and is catalyzed by editosomes containing the essential enzyme REL1, which has no close human homologs and is thus a potential drug target.
(2) Molecular dynamics simulations were used to virtually screen compounds, identifying three initial inhibitors of REL1 with low micromolar IC50 values.
(3) Additional screening of databases identified 588 related compounds, of which 25 top candidates inhibited REL1 in biochemical assays with IC50 values of 1-10 μM. Efforts are underway to improve activity and selectivity.
This document provides information for delegates attending the 50th Spring Meeting of the British Society for Parasitology (BSP) in Glasgow, Scotland. It includes details about registration, venue facilities, the scientific program, social events, and logistics. The meeting will feature keynote talks on malaria and schistosomiasis to mark 50 years of the BSP. Other highlights include a plenary lecture by Lord May, the Wright Medal lecture by Professor Mark Taylor, and a debate on the future of parasitic disease control. Social events include a reception at Glasgow City Chambers and the conference dinner at Kelvingrove Art Gallery and Museum.
The use of Nauplii and metanauplii artemia in aquaculture (brine shrimp).pptxMAGOTI ERNEST
Although Artemia has been known to man for centuries, its use as a food for the culture of larval organisms apparently began only in the 1930s, when several investigators found that it made an excellent food for newly hatched fish larvae (Litvinenko et al., 2023). As aquaculture developed in the 1960s and ‘70s, the use of Artemia also became more widespread, due both to its convenience and to its nutritional value for larval organisms (Arenas-Pardo et al., 2024). The fact that Artemia dormant cysts can be stored for long periods in cans, and then used as an off-the-shelf food requiring only 24 h of incubation makes them the most convenient, least labor-intensive, live food available for aquaculture (Sorgeloos & Roubach, 2021). The nutritional value of Artemia, especially for marine organisms, is not constant, but varies both geographically and temporally. During the last decade, however, both the causes of Artemia nutritional variability and methods to improve poorquality Artemia have been identified (Loufi et al., 2024).
Brine shrimp (Artemia spp.) are used in marine aquaculture worldwide. Annually, more than 2,000 metric tons of dry cysts are used for cultivation of fish, crustacean, and shellfish larva. Brine shrimp are important to aquaculture because newly hatched brine shrimp nauplii (larvae) provide a food source for many fish fry (Mozanzadeh et al., 2021). Culture and harvesting of brine shrimp eggs represents another aspect of the aquaculture industry. Nauplii and metanauplii of Artemia, commonly known as brine shrimp, play a crucial role in aquaculture due to their nutritional value and suitability as live feed for many aquatic species, particularly in larval stages (Sorgeloos & Roubach, 2021).
The ability to recreate computational results with minimal effort and actionable metrics provides a solid foundation for scientific research and software development. When people can replicate an analysis at the touch of a button using open-source software, open data, and methods to assess and compare proposals, it significantly eases verification of results, engagement with a diverse range of contributors, and progress. However, we have yet to fully achieve this; there are still many sociotechnical frictions.
Inspired by David Donoho's vision, this talk aims to revisit the three crucial pillars of frictionless reproducibility (data sharing, code sharing, and competitive challenges) with the perspective of deep software variability.
Our observation is that multiple layers — hardware, operating systems, third-party libraries, software versions, input data, compile-time options, and parameters — are subject to variability that exacerbates frictions but is also essential for achieving robust, generalizable results and fostering innovation. I will first review the literature, providing evidence of how the complex variability interactions across these layers affect qualitative and quantitative software properties, thereby complicating the reproduction and replication of scientific studies in various fields.
I will then present some software engineering and AI techniques that can support the strategic exploration of variability spaces. These include the use of abstractions and models (e.g., feature models), sampling strategies (e.g., uniform, random), cost-effective measurements (e.g., incremental build of software configurations), and dimensionality reduction methods (e.g., transfer learning, feature selection, software debloating).
I will finally argue that deep variability is both the problem and solution of frictionless reproducibility, calling the software science community to develop new methods and tools to manage variability and foster reproducibility in software systems.
Exposé invité Journées Nationales du GDR GPL 2024
Immersive Learning That Works: Research Grounding and Paths ForwardLeonel Morgado
We will metaverse into the essence of immersive learning, into its three dimensions and conceptual models. This approach encompasses elements from teaching methodologies to social involvement, through organizational concerns and technologies. Challenging the perception of learning as knowledge transfer, we introduce a 'Uses, Practices & Strategies' model operationalized by the 'Immersive Learning Brain' and ‘Immersion Cube’ frameworks. This approach offers a comprehensive guide through the intricacies of immersive educational experiences and spotlighting research frontiers, along the immersion dimensions of system, narrative, and agency. Our discourse extends to stakeholders beyond the academic sphere, addressing the interests of technologists, instructional designers, and policymakers. We span various contexts, from formal education to organizational transformation to the new horizon of an AI-pervasive society. This keynote aims to unite the iLRN community in a collaborative journey towards a future where immersive learning research and practice coalesce, paving the way for innovative educational research and practice landscapes.
Or: Beyond linear.
Abstract: Equivariant neural networks are neural networks that incorporate symmetries. The nonlinear activation functions in these networks result in interesting nonlinear equivariant maps between simple representations, and motivate the key player of this talk: piecewise linear representation theory.
Disclaimer: No one is perfect, so please mind that there might be mistakes and typos.
dtubbenhauer@gmail.com
Corrected slides: dtubbenhauer.com/talks.html
ESPP presentation to EU Waste Water Network, 4th June 2024 “EU policies driving nutrient removal and recycling
and the revised UWWTD (Urban Waste Water Treatment Directive)”
EWOCS-I: The catalog of X-ray sources in Westerlund 1 from the Extended Weste...Sérgio Sacani
Context. With a mass exceeding several 104 M⊙ and a rich and dense population of massive stars, supermassive young star clusters
represent the most massive star-forming environment that is dominated by the feedback from massive stars and gravitational interactions
among stars.
Aims. In this paper we present the Extended Westerlund 1 and 2 Open Clusters Survey (EWOCS) project, which aims to investigate
the influence of the starburst environment on the formation of stars and planets, and on the evolution of both low and high mass stars.
The primary targets of this project are Westerlund 1 and 2, the closest supermassive star clusters to the Sun.
Methods. The project is based primarily on recent observations conducted with the Chandra and JWST observatories. Specifically,
the Chandra survey of Westerlund 1 consists of 36 new ACIS-I observations, nearly co-pointed, for a total exposure time of 1 Msec.
Additionally, we included 8 archival Chandra/ACIS-S observations. This paper presents the resulting catalog of X-ray sources within
and around Westerlund 1. Sources were detected by combining various existing methods, and photon extraction and source validation
were carried out using the ACIS-Extract software.
Results. The EWOCS X-ray catalog comprises 5963 validated sources out of the 9420 initially provided to ACIS-Extract, reaching a
photon flux threshold of approximately 2 × 10−8 photons cm−2
s
−1
. The X-ray sources exhibit a highly concentrated spatial distribution,
with 1075 sources located within the central 1 arcmin. We have successfully detected X-ray emissions from 126 out of the 166 known
massive stars of the cluster, and we have collected over 71 000 photons from the magnetar CXO J164710.20-455217.
Current Ms word generated power point presentation covers major details about the micronuclei test. It's significance and assays to conduct it. It is used to detect the micronuclei formation inside the cells of nearly every multicellular organism. It's formation takes place during chromosomal sepration at metaphase.
The technology uses reclaimed CO₂ as the dyeing medium in a closed loop process. When pressurized, CO₂ becomes supercritical (SC-CO₂). In this state CO₂ has a very high solvent power, allowing the dye to dissolve easily.
1. Repeat Km evaluationRepeat Km evaluation
ImmediatelyImmediately
Near futureNear future
Titrate K+ and Mg 2+ ions in 10 x Adenylation (ligation) bufferTitrate K+ and Mg 2+ ions in 10 x Adenylation (ligation) buffer
Substitute Tris for Hepes (@pH 7.9) in bufferSubstitute Tris for Hepes (@pH 7.9) in buffer
SubstituteSubstitute ββ ME for DTTME for DTT
Upon completion repeat Z’ factor assay with reference to intra and interUpon completion repeat Z’ factor assay with reference to intra and inter
plate variabilityplate variability
Actually….Actually….
Further investigated chemical denaturation conditionsFurther investigated chemical denaturation conditions
Revisited L1 time curve kinetics to verify current #001 prep activity/kineticsRevisited L1 time curve kinetics to verify current #001 prep activity/kinetics
Continued to optimise induction conditions as a prelude to a 2Continued to optimise induction conditions as a prelude to a 2ndnd
roundround
(#002) of L1 purification by affinity (His tag) LC(#002) of L1 purification by affinity (His tag) LC
Anticipated….Anticipated….
2. Specific Signal Versus L1-A2 Batch (#001)
#001_1
#001_2
#001_3
#001_4
#001_5
#001_6
#001_6_6/7
#001_6_8
#001_6_9
#001_6_10
T4RNALigase2
No L1
0.0
10000.0
20000.0
30000.0
40000.0
50000.0
60000.0
#001_1-6 + T4 RNA ligase 2 + No L1 control
Batch #_#001
AFU
S/B ~ 8 XS/B ~ 8 X
Assay # 008: 001-L1 ~ 3 months -80Assay # 008: 001-L1 ~ 3 months -80oo
CC
Gain =47Gain =47
3. Comparing FRET output from ligated products versus [ ATP ] versus L1 mass/rxn
NoL1
100ng
250ng
500ng
NoL1_5uMATP
100ng
250ng
500ng
0.0E+00
5.0E+03
1.0E+04
1.5E+04
2.0E+04
2.5E+04
3.0E+04
#2 + #1 No L1
AFU
3.1x 5.4x 6.0x
5µM
ATP
5µM
ATP
5µM
ATP
40µM
ATP
40µM
ATP
40µM
ATP
Fresh #001-6
3 month #001-6
Ligation less efficient with lower [ATP]Ligation less efficient with lower [ATP]
Consequently specific signal lessConsequently specific signal less
However, because background is exceptionally low, S/B – the real hall mark of assay quality - isHowever, because background is exceptionally low, S/B – the real hall mark of assay quality - is
actually higher ?!! Why ? Is the use of fresh DEPC water versus ‘old’ Sigma water at least part ofactually higher ?!! Why ? Is the use of fresh DEPC water versus ‘old’ Sigma water at least part of
the answer ?the answer ?
4. No significant appreciation in L1 activity over time course duration suggesting a loss of intrinsic activity
This precludes linear order reactions and inhibition studies
Thus new L1 must now be purified !!
5. Time course analysis of 3 L1 (#001) preps @ 111.25ng & 222.5ng versus T4 RNA ligase 2 (#033)
versus Assay #004B
T4 RNA Ligase 2,
40000.25T4 RNA Ligase 2,
37506.3
No L1,
2328.75
No L1,
2467.75
1ug/ul BSA
1ug/ul BSA
0
10000
20000
30000
40000
50000
60000
70000
0min 10min 20min 40min 60min 120min
Ligation time
AFU
#001-2-1_211010_111.25ng
#001-2-1_211010_222.5ng
#001-6_141010_111.25ng
#001-6_141010_222.5ng
#001-6-8_280610(dil)_112.5ng
#001-6-8(dil)_280610_222.5ng
T4 RNA Ligase 2
No L1
#001-6_2/5_Assay #004B_111.25ng
#001-6-2/5_#004B_1ug/ul BSA
Gain = 51
Blue = Fresh:
6 months @ -80o
C
Red = 1 week @
-20o
C
Violet = 4 months
(prediluted) @ -
20o
C
#033
2 months @ -80o
C
#004B
#033
L1 Now 6 months
No significant appreciation in L1 activity over time course duration (#033) suggesting a loss of intrinsic
activity
This pertains to all preps tested (~ 6 months old) irrespective of storage conditions
Curiously, the prep exhibiting least activity was newly thawed, i.e. ~ 6 months @ -80o
C ??!!
6. #1#1 #2#2 #3#3 #4#4
50KD
40KD
30KD
20KD
PP PP PP P?P?SS PP PP PP P?P?SS SS SS SS SS S?S? S?S?
+ + + +- - - -
Catalytic domainCatalytic domain
REL1REL1
Specific and high level expression of L1 (catalytic REL 1 domain) in pellet
No obvious sign of soluble protein !
Is this due to a lack of A2 (N-terminal TEV His tag) rendering L1 soluble ?
+/+/-- 0.5mM IPTG0.5mM IPTGWestern blotting
with:
α REL 1Ab
α His tag Ab
7. ++ ++-- -- ++ ++-- --
Bug buster/PBS + lysozyme/
Benzonase + 1% Triton X-100+ 1% Triton X-100
Buffer A + lysozyme/
Benzonase
+/- 0.5mM
IPTG
Buffer A + lysozyme/
Benzonase + 1% Triton+ 1% Triton
X-100X-100
Bug buster/PBS + lysozyme/
Benzonase
Clone #1_2Clone #1_2 Clone #5_1Clone #5_1
PPPP PP PP PPSS SS
Clone #1_3Clone #1_3 Clone #5_2Clone #5_2
SS SSPP PP PP PPSSSS SS SS SS
Cultures grown to an initial OD600 of 0.5_0.6 at 37o
C for 1.5 hours by seeding 2ml of LB with 25ul of overnight
Induction achieved by splitting 2ml culture into 2 x 1ml aliquots and then adding 6.25ul of 1/10 0.8M IPTG to one such
aliquot (2nd
= un induced control) followed by growth @18o
C for 20-21 hours
Like original prep (i.e. Initial OD600 0.8 followed by induction for 20 hours @ 20o
C in presence of 0.5mM IPTG) = #1_1
REL 1 remains in pelleted fraction
Similarly, TEV cleavable N terminal His tagged A2 remains associated with pellet
5050
4040
3030
2020
1515
1010
3.53.5
L1
51-469
A2
56-176
4-12% Bis Tris Novex gel
1 x MES
200V 45 minutes
Pellet
#1_1 (+)
Supernatant
#1_1(+)
8. 50KD
40KD
30KD
20KD
PP PP PP PPSS PP PP PP PPSS SS SS SS SS SS SS
Catalytic domainCatalytic domain
REL1 (R1)REL1 (R1)
Chaperone (A2):Chaperone (A2):
N Terminal TEVN Terminal TEV
His TagHis Tag
#1#1 #2#2 #4#4 HS- #1HS- #1
+ + + +- - -- +/+/-- 0.5mM IPTG0.5mM IPTG
Heat shock @ 42o
C 10min; 37o
C 20 min; ice 30 min clearly solubilised some L1
9. 50KD
40KD
30KD
20KD
PP PP PP P+P+
SS
P+P+ P+P+ P+P+ PP
S+S+ SS S+S+ SS S+S+ SS S+S+
Catalytic domainCatalytic domain
REL1 (R1)REL1 (R1)
Chaperone (A2):Chaperone (A2):
N Terminal TEVN Terminal TEV
His TagHis Tag
+ 0.1mM+ 0.1mM + 0.5mM+ 0.5mM + 2.0mM+ 2.0mM No IPTGNo IPTG
Key:Key: P = pellet P+ = + heat shock
S = Spt. S+ = + heat shock
Less IPTG might actually be more effective at rendering L1
more soluble
In common with induction #5 heat shock may further promote
this trend
10. The effect of denaturation regimen +/- g R#1 Annealing
gR#1_noheat
gR#1_noheat
gR#1_noheat
gR#1_noheat
gR#1+heat
gR#1+heat
gR#1+heat
gR#1+heat
NogR#1_noheat
NogR#1_noheat
NogR#1_noheat
NogR#1_noheat
NogR#1_heat
NogR#1_heat
NogR#1_heat
NogR#1_heat
0
5000
10000
15000
20000
25000
30000
#139_ No #139 80 % Form 80 % DMSO
Denaturation conditions
AFU
g R#1_no heat
gR#1 + heat
No gR#1_no heat
No gR#1_heat
In the presence of g R #1 irrespective of denaturation
consistently higher signal
In the absence of g R#1 signal +/- heat approx the same
In the absence of #139 heat denaturation less effective
For both formamide and (especially DMSO) significant signal
quenching apparent
This quenching was confirmed by similar magnitude of effect
associated with signal from double labelled oligo control
Optimal conditions in terms of S/B = + g R #1 + #139 = 4.5
EDTA + #139EDTA + #139
5mM Mg; 25mM K; no #139 = 1 x Adenylation buffer5mM Mg; 25mM K; no #139 = 1 x Adenylation buffer
Gain = 41Gain = 41
Can 1 x adenylation buffer potentiate specific signal or is this an artifact of high [mg] &/or no #139/EDTA ?
11. Manifest quenching of dual labelled Oligo by Formamide Titrants
#139STOP
#139STOP
#139STOP
25%
25%
25%
50%
50%50%
60%
60%
60%
70%
70%
70%
80%
80%80%
90%
90%
90%
100%
100%
100%
0
5000
10000
15000
20000
25000
30000
35000
40000
FRET FAM Cy5
Signal Output
AFU
#139 STOP
25%
50%
60%
70%
80%
90%
100%
Formamide @ all [ ] appears to be quenching FRET signal relative to #139 stopFormamide @ all [ ] appears to be quenching FRET signal relative to #139 stop
This can be attributed to quencing of FAM as Cy5 output comparable irrespective of conditionsThis can be attributed to quencing of FAM as Cy5 output comparable irrespective of conditions
12. Manifest quenching of dual labelled Oligo by DMSO Titrants > 50%
#139STOP
#139STOP
#139STOP
25%
25%
25%
50%
50%
50%
60%
60%
60%
70%
70%
70%
80%
80%
80%
90%
90%
90%
100%
100%
100%
0
5000
10000
15000
20000
25000
30000
35000
40000
45000
50000
FRET FAM Cy5
Signal Output
AFU
#139 STOP
25%
50%
60%
70%
80%
90%
100%
Above 50% [ ] DMSO appears to be quenching FRET signal relative to #139 stopAbove 50% [ ] DMSO appears to be quenching FRET signal relative to #139 stop
Below 50% DMSO FRET signal comparable to #139 stopBelow 50% DMSO FRET signal comparable to #139 stop
HoweverHowever ‘apparent’ parity of FRET may in fact belie quenched FAM output coupled to enhanced‘apparent’ parity of FRET may in fact belie quenched FAM output coupled to enhanced
Cy5Cy5
13. #139 stop signal magnitude plus heat almost equivalent to scaffold without g R#1 implying#139 stop signal magnitude plus heat almost equivalent to scaffold without g R#1 implying
efficient heat denaturationefficient heat denaturation
In the absence of heat some denaturation apparent for 50% DMSOIn the absence of heat some denaturation apparent for 50% DMSO
Apparently, denaturation by heat + DMSO less efficient than for #139 stop + heatApparently, denaturation by heat + DMSO less efficient than for #139 stop + heat
Both in the presence and absence of heat, DMSO mixtures should have included (molar excess)Both in the presence and absence of heat, DMSO mixtures should have included (molar excess)
#139 to preclude renaturation#139 to preclude renaturation
Thus, denaturation in this context undermined (potentially) by absence of #139: Include next time !Thus, denaturation in this context undermined (potentially) by absence of #139: Include next time !
FRETFRET
14. Regarding FAM signal from intact (+ g R#1) scaffold, #139 + heat yielded a higher output than in theRegarding FAM signal from intact (+ g R#1) scaffold, #139 + heat yielded a higher output than in the
absence of heat; This cab be attributed to quenching of signal by FRET in the latterabsence of heat; This cab be attributed to quenching of signal by FRET in the latter
In the case of DMSO, signal yielded by intact (+g R#1) scaffold consistently lower than for scaffoldIn the case of DMSO, signal yielded by intact (+g R#1) scaffold consistently lower than for scaffold
+ #139 stop buffer: Is this due to FRET quenching augmented by chemical quenching of FAM+ #139 stop buffer: Is this due to FRET quenching augmented by chemical quenching of FAM
molecules ?molecules ?
In common with #139 stop quenching of FAM by FRET in the context of DMSO indicated by augmentedIn common with #139 stop quenching of FAM by FRET in the context of DMSO indicated by augmented
signal in the presence of heat and moreover absence of gR #1 where scaffold formation is precludedsignal in the presence of heat and moreover absence of gR #1 where scaffold formation is precluded
FAMFAM
15. Cy5Cy5
Regarding Cy 5 output, consistent signal enhancement caused by DMSO (compared to #139 stop)Regarding Cy 5 output, consistent signal enhancement caused by DMSO (compared to #139 stop)
irrespective of heat application or not; inclusion of g R#1 or notirrespective of heat application or not; inclusion of g R#1 or not
This suggests an intrinsic effect on the Cy5 dye molecule itself by DMSOThis suggests an intrinsic effect on the Cy5 dye molecule itself by DMSO
16. Signal potentiation by adenylation buffer can be attributed to Mg & HEPES respectivelySignal potentiation by adenylation buffer can be attributed to Mg & HEPES respectively
Thus, could stop supplemented buffer supplemented with extra HEPES boost S/B ?Thus, could stop supplemented buffer supplemented with extra HEPES boost S/B ?
Caveat:Caveat: Will this interfere with efficient denaturation ?Will this interfere with efficient denaturation ?
17. ExtraExtra Hepes buffer does not interfere with efficient denaturationHepes buffer does not interfere with efficient denaturation
However neither does it augment S/BHowever neither does it augment S/B
Not suprising !!
Omitting #139 & EDTA culminates in less efficient denaturationOmitting #139 & EDTA culminates in less efficient denaturation
0.1X0.1X 1X1X 1X1X
0.1X0.1X
- #139- #139
19. Signal from both molarities of adenylation bufferSignal from both molarities of adenylation buffer
much higher than Fam-Cy5 oligo diluted in watermuch higher than Fam-Cy5 oligo diluted in water
Signal from 0.1 x strength and 1 x strength theSignal from 0.1 x strength and 1 x strength the
samesame
How does this signal compare with black 96 wellHow does this signal compare with black 96 well
plates ?plates ?
What components of the buffer augment signalWhat components of the buffer augment signal
compared with water alone and could these becompared with water alone and could these be
manipulated (in ‘stop buffer’) to boost S/B ?manipulated (in ‘stop buffer’) to boost S/B ?
20. The effect of diluent on signal emanating from 1:2 Serial Titration of Fam-Cy5 Double labelled Oligo: 96 well White
Greiner plate
y = -0.9156x + 12.584
R
2
= 0.9979
y = -0.9414x + 16.776
R
2
= 0.9995
y = -0.8545x + 16.365
R
2
= 0.9971
0
2
4
6
8
10
12
14
16
18
500 250 125 62.5 31.25 15.625 7.8125 3.90625 1.953125
Conc FAM-Cy5 Oligo_nM
Log(2)Specificsignal
Water Diluent
0.1 x Adenylation buffer diluent
1 x Adenylation buffer diluent
Log 2 water trend line
Log 2 Trend line 1 x adenylation
Log 2 0.1 x Adenylation trend line
Red = water
Yellow = 0.1 x
Pink = 1 x
21. S/B determined by dividing specific signal byS/B determined by dividing specific signal by
buffer/water without dye labelled Oligobuffer/water without dye labelled Oligo
S/B mirror previous absolute signal resultsS/B mirror previous absolute signal results
22. Signal associated with white Greiner plates falls toSignal associated with white Greiner plates falls to
background with diminished oligo less rapidly than forbackground with diminished oligo less rapidly than for
black plates, i.e. white plate sensitivity higherblack plates, i.e. white plate sensitivity higher
S/B for black plates could not be computed on account ofS/B for black plates could not be computed on account of
high gain, consequent higher background and –ive wellshigh gain, consequent higher background and –ive wells
below this backgroundbelow this background
Assay [ oligo }: should gain be adjusted to this point
for meaningful S/B determination ?
23. Sensitivity of white Greiner plate muchSensitivity of white Greiner plate much
higher than black Greiner platehigher than black Greiner plate
Consequently, gain can be set much lowerConsequently, gain can be set much lower
Thus, negative well higher than blank wellThus, negative well higher than blank well
This enables meaningful Signal to backgroundThis enables meaningful Signal to background
to be computedto be computed
Sensitivity of black plates lessSensitivity of black plates less
Consequently, gain adjustmentConsequently, gain adjustment
results in back ground signal >results in back ground signal >
blank wellsblank wells
This precludes meaningful S/BThis precludes meaningful S/B
calculationcalculation
However, should gain be adjustedHowever, should gain be adjusted
to conc. range corresponding toto conc. range corresponding to
actual [ dye labelled oligo ] ?actual [ dye labelled oligo ] ?
24. Make 2 litre induction of L1-A2 using 0.1mM IPTG + heat shockMake 2 litre induction of L1-A2 using 0.1mM IPTG + heat shock
Freeze away samples in thin walled 0.2ml tubes +/- cryoprotectants, viz.Freeze away samples in thin walled 0.2ml tubes +/- cryoprotectants, viz.
L-arginine & sulfobetainesL-arginine & sulfobetaines
Quantify Licor gel with heat shock/IPTG titration and rerun mass equivalentQuantify Licor gel with heat shock/IPTG titration and rerun mass equivalent
samples for verification/quantitation of soluble L1 by Western blottingsamples for verification/quantitation of soluble L1 by Western blotting
Repeat 50% DMSO denaturation + #139 and evaluate in context of mild temp.Repeat 50% DMSO denaturation + #139 and evaluate in context of mild temp.
gradient (viz. RT-60gradient (viz. RT-60oo
C)C)
In addition try denaturation with 6M urea & DMSO + 1.5M (deionised) glyoxalIn addition try denaturation with 6M urea & DMSO + 1.5M (deionised) glyoxal
Before xmasBefore xmas
Near futureNear future
Titrate K+ and Mg 2+ ions in 10 x Adenylation (ligation) bufferTitrate K+ and Mg 2+ ions in 10 x Adenylation (ligation) buffer
Substitute Tris for Hepes (@pH 7.9) in bufferSubstitute Tris for Hepes (@pH 7.9) in buffer
SubstituteSubstitute ββ ME for DTTME for DTT
Perform Triton X-100 titration in ligation reactions & compare withPerform Triton X-100 titration in ligation reactions & compare with Brij-35Brij-35®®
Upon completion repeat Z’ factor assay with reference to intra and interUpon completion repeat Z’ factor assay with reference to intra and inter
plate variabilityplate variability
Accurately determine Km [ATP] in context ofAccurately determine Km [ATP] in context of E ColiE Coli L1 prep !!L1 prep !!