Daekitech

Abstract

pENTR223.1 plasmid -LC is a gateway cloning vector for bacterial expression. The vector is 3375 bp in size and contains the pBR322 origin of replication. This vector carries two genes, ccdB and CmR, in the region that will be replaced by cloned DNA segments. The ccdB gene (DNA gyrase inhibitor) provides strong negative selection against vector molecules that retain this region, while CmR (chloramphenicol resistance gene) provides positive selection for the propagation of the vector. The sequencing primers are T7, M13 and M13reverse. pENTR223.1-LC is resistant to spectinomycin up to 100 ug/ml.

Bottom

Valuable collections of clones encoding the complete ORFeomes for some model organisms have been built following the completion of their genome sequencing projects. These libraries are based on Gateway cloning technology, which facilitates the study of protein function by simplifying the subcloning of open reading frames (ORFs) into any suitable target vector.

The expression of proteins of interest as fusions with functional modules is a frequent approach in their initial functional characterization. A limited number of Gateway target expression vectors allow the construction of fusion proteins from ORFeome-derived sequences but are restricted to the possibilities offered by their built-in functional modules and predefined model organism specificity. Therefore, the availability of cloning systems that overcome these limitations would be highly advantageous.

Construction of pEF5/FRT/V5-DEST-R4-R3

Adaptation of pEF5/FRT/V5-DEST for MultiSite Gateway cloning was carried out as described by Magnani et al, with modifications. Briefly, an LR recombination reaction was carried out at 25 °C for 16 h between plasmids pEF5/FRT/V5-DEST_CamS (see above) and pDONR221-R4-R3. Importantly, the attR4/attR3 sites in pDONR221-R4-R3 cannot recombine with the juxtaposed attL1/attL2 sites. After transformation of the reaction into code Survival™ 2 T1R competent cells, cultures were plated on LB/agar plates containing ampicillin and chloramphenicol to select for colonies harbouring the recombined pEF5/FRT/V5-DEST plasmid.

In this way, the standard Gateway cassette flanked by attR1/attR2 in pEF5/FRT/V5-DEST was replaced by the Gateway MultiSite cassette flanked by attR4/attR3, obtaining pEF5/FRT/V5-DEST-R4-R3, which was ready for a MultiSite LR recombination cloning reaction with the three modules that would constitute the chosen fusion protein.

Molecular biology

The PCR reactions were carried out with AccuPrime Pfx SuperMix (Life Technologies) as indicated by the manufacturer. Plasmids pDONR and the cloning vector PDT-R4-R3 were obtained as part of the MultiSite Gateway Cloning Kit (Life Technologies Catalog No. 12537023). The pEF5/FRT/V5-DEST destination expression vector was obtained from Life Technologies (catalogue #V6020-20). This is a vector for cloning and expressing proteins in FLP-In™ isogenic cell lines.

The expression of cloned proteins is driven by the human elongation factor 1α promoter located upstream of the Gateway cassette. The vector has an FRT recombination site that mediates FLP recombinase-directed integration of the vector into a unique homologous FRT site in the genome of FLP-In™ cell lines, and the hygromycin resistance gene acts as a selectable marker for integration. However, it behaves like any other expression vector in transient transfection experiments.

BP and LR recombination reactions were carried out with BP clonase II and LR Clonase II Plus enzyme mixtures, respectively (Life Technologies), following the manufacturer’s instructions. Reactions were stopped by the addition of proteinase K and incubation at 37°C for 10 min. All competent strains of E. coli were obtained from Life Technologies and used in a one-shot format for plasmid transformation. LB medium was supplemented where indicated with selection antibiotics at the following concentrations: ampicillin (100 μg/mL), kanamycin (50 μg/mL), chloramphenicol (50 μg/mL).

Results

We present a versatile cloning toolkit for constructing fully customizable three-part fusion proteins based on the MultiSite Gateway Cloning System. The components of the fusion protein are encoded on the three integral plasmids of the kit. These can be recombined with any purposely designed target vector using a heterologous promoter external to the Gateway cassette, leading to in-frame cloning of an ORF of interest flanked by two functional modules.

Unlike previous systems, a third part is available for peptide encoding as it no longer needs to contain a promoter, resulting in a greater number of possible fusion combinations. We have constructed the component plasmids of the kit and demonstrated their functionality by providing proof-of-principle data on the expression of prototype fluorescent fusions in transiently transfected cells.

Conclusions

We have developed a set of tools to create fusion proteins with custom N- and C-term modules from Gateway input clones encoding ORFs of interest. Importantly, our method allows input clones obtained from ORFeome collections to be used without prior modification. Using this technology, any existing Gateway target expression vector with its template-specific properties could be easily adapted to express fusion proteins.

Abstract

A series of plasmid vectors containing the multiple cloning site (MCS7) of M13mp7 have been constructed. In one of these vectors, a kanamycin resistance marker has been inserted into the centre of the symmetrical MCS7 to produce a restriction site mobilizer (RSM) element. The drug resistance marker can be excised from this vector with any of the restriction enzymes that recognize a site in the RSM flanking sequences to generate an RSM with various sticky ends or blunt ends. These fragments can be used for insertional mutagenesis of any target molecule with compatible restriction sites.

Insertion mutants are selected for their resistance to kanamycin. When the drug resistance marker is removed with PstI, a small in-frame insert can be generated. In addition, two new MCS have been formed that have unique restriction sites by disrupting the symmetrical structure of MCS7. The resulting plasmids pUC8 and pUC9 allow separate double-digested restriction fragments to be cloned in both orientations relative to the lac promoter. The terminal sequences of any DNA cloned into these plasmids can be characterized using the M13 universal primers.

pUC plasmid vectors are used as cloning vectors and belong to the pUC series (named after the place of their initial preparation, ie, the University of California). These plasmids are 2700 bp long and contain

• Ampicillin resistance gene
• Origin of replication derived from pBR322,
• lacZ gene derived from E. coli. Also found within the lac region is a polylinker sequence that has unique restriction sites (identical to those found in phage M13), and
• Multiple Cloning Sites (MCS).

These plasmids, when transformed into an appropriate E.coli strain having lac (eg, JM103, JMI09), and grown in the presence of IPTG (isopropyl thiogalactoside, which behaves like lactose and induces lactose synthesis). of the enzyme galactosidase f3) and X-gal (substrate for the enzyme), will give rise to white or clear colonies.

On the other hand, the pUC that does not have inserts and is transformed into bacteria will have an active lacZ gene and will therefore produce blue colonies, which will make it possible to identify the colonies that have the pUC vector with cloned DNA segments.

As discussed above, in pBR322 and pBR327, the DNA is inserted into a site located in one of the two antibiotic resistance genes, thereby inactivating one of the two resistance genes. The insert-carrying plasmid can be selected for its ability to grow in a medium containing only one of the two antibiotics and for its inability to grow in a medium containing both antibiotics.

On the other hand, plasmids that do not carry an insert grow in media containing one or both antibiotics. Thus, the presence of the lacZ gene in pUC and ampicillin and tetracycline resistance genes in pBR322 and pBR327 allow selection of E. coli colonies transformed with plasmids carrying the desired foreign cloned DNA segment.

pUC19 is a commonly used high copy cloning vector. The vector encodes the N-terminal fragment of β-galactosidase (Lanza), which allows detection of blue/white colonies (i.e. complement a), as well as a pUC origin of replication and an ampicillin resistance gene that allow propagation and selection. in E. coli.

An important feature of pUC plasmids is the blue/white colony screen to detect recombinant plasmids. This selection is based on the inactivation of the Lanza peptide of beta-galactosidase, which is expressed by the vector. Cloning vectors belonging to the pUC family are available in pairs with reverse orders of restriction sites relative to the lacZ promoter. pUC8 and PUC9 form one such pair. Other similar pairs include pUC12 and pUCl3 or pUC18 and pUC19.

Disadvantages of pUC vectors

The basis of genomic sequence analysis is the large-scale cloning and sequencing of shotgun plasmid libraries obtained by assembling sequences from the vast majority of clones.

• Breaches in shotgun libraries and unclonable DNA fragments are quite common. DNA is characterized by high AT content, strong secondary structure, open reading frames, or cis-acting functions (eg, transcriptional promoters or origins of replication).
• In some cases, most notably in AT-rich DNA, the reasons for the difficulty in cloning are not well defined.

Abstract

Creative Biogene is a biotechnology company specializing in custom plasmid DNA production services. With years of experience servicing plasmid DNA production, Creative Biogene can offer custom plasmid services on microgram to gram scales using Creative Biogene’s unique and proprietary process. In addition, Creative Biogene can provide you with plasmids of various quality grades, including research-grade, endotoxin-free grade, and GMP grade plasmid DNA. Creative Biogene’s goal is to provide you with the most affordable high-quality plasmid DNA manufacturing services to ensure your satisfaction in a timely and professional manner.

Customized Plasmids are small DNA molecules that are physically separate from chromosomal DNA and can replicate independently. They are most commonly found in bacteria as small circular double-stranded DNA molecules. Today, plasmid DNAs serve as important tools in genetic engineering. They are commonly used in genetics and biotechnology laboratories, where they are used to clone and amplify (make many copies of) or express particular genes. Creative Biogene can provide you with high-quality plasmid DNA production services. You can select scales and grades of production quality to best meet your specific needs.

Creative Biogene can provide you with plasmids of multiple grades of quality. You can select the production-grade that best suits your needs.

• Research Grade – For typical molecular biology research including recloning, sequencing, PCR, and protein manufacturing.
• Endotoxin-free grade – recommended for effective cell transfection, genetic protein and vaccine manufacturing, and animal studies
• GMP Grade: GMP grade plasmid DNA can be used for DNA vaccines, gene therapy, or ex vivo applications.

Creative Biogene offers customized plasmid DNA production services for your scientific research as follows:

• Selection of the host strain and growth conditions.
• High cell density fermentation.
• Cell harvesting, lysis and purification of plasmids.
• Wide range of quality control tests, including genomic RNA/DNA/protein contamination test, endotoxin (lipopolysaccharide) analysis, restriction profile analysis, DNA sequencing, OD260/280 readings, etc.

Product name

tissue transglutaminase IgA, tTG-IgA, ELISA kit

Full product name

Human Tissue Transglutaminase IgA, tTG-IgA ELISA kit

Product gene name

tTG-IgA ELISA kit

Species reactivity: Human

Specificity

No significant cross-reactivity or interference is observed between this analyte and analogues.

Samples

Human body fluids and tissue homogenates

Test type: Quantitative sandwich

Detection range: 31.2pg/ml-1000pg/ml

Sensitivity: 5.0pg/mL.

Intra-assay precision

Intra-assay CV (%) is less than 15%

Inter-assay precision

The inter-assay CV (%) is less than 15%. [CV(%) = SD/mean ×100]. All CV% should be compared by concentration, not by OD values.

Preparation and Storage

Store all reagents at 2-8 degrees C

Product note

Information selected from the online data sheet is extracted from bioinformatics databases, sometimes resulting in the ambiguous or non-relevant product information. It is the customer’s responsibility to review, verify and evaluate the information to ensure it matches their requirements prior to purchasing the kit. Our ELISA Kit assays are dynamic research tools and may be updated and improved from time to time.

If the format of this assay is important to you, please request the current manual or contact our technical support team with a presales inquiry before placing an order. We will confirm the current trial details. We cannot guarantee that the sample manual posted online is the most up-to-date manual, it is intended to serve as an example only. Consult the instructions for use provided with the assay kit for precise details.

Other notes

On occasion, small volumes of tTG-IgA ELISA kit vials may become trapped in the product vial seal during shipping and storage. If necessary, briefly spin the vial in a tabletop centrifuge to dislodge any liquid in the vial cap. Certain products may require dry ice shipping and an additional dry ice fee may apply.

Search terms for the purchase of tTG-IgA

MBS9302975 is a ready-to-use microwell and strip plate ELISA (Enzyme-Linked Immunosorbent Assay) Kit for testing the presence of Tissue Transglutaminase IgA, tTG-IgA ELISA Kit target analytes in biological samples. The concentration gradients of the kit standards or positive controls generate a theoretical detection range of the kit in biological research samples containing tTG-IgA.

The analytical biochemical ELISA technique of the MBS9302975 kit is based on tTG-IgA antibody-tTG-IgA antigen (immunosorbent) interactions and an HRP colourimetric detection system to detect tTG-IgA antigen targets in samples. The ELISA Kit is designed to detect native, non-recombinant tTG-IgA. Appropriate sample types may include undiluted body fluids and/or tissue homogenates, secretions. Quality control assays that evaluated reproducibility identified intra-assay CV (%) and inter-assay CV (%).

Product related information for tTG-IgA ELISA kit

Background/Introduction: This quantitative sandwich ELISA kit is for laboratory reagents/research use only! This kit is intended to be used to determine the level of TSST-1 (hereinafter referred to as “analyte”) in undiluted original human body fluids and tissue homogenates. This kit is NOT suitable for testing non-biological sources of substances.

Precautions

All MyBioSource products are for scientific laboratory research purposes and not for diagnostic, therapeutic, prophylactic, or in vivo use. Through your purchase, you expressly represent and warrant to MyBioSource that you will properly test and use any Product purchased from MyBioSource in accordance with industry standards. MyBioSource and its authorized distributors reserve the right to refuse to process any order where we reasonably believe the intended use will not meet our acceptable guidelines.

Disclaimer

While every effort has been made to ensure the accuracy of the information provided in this datasheet, MyBioSource shall not be responsible for any omissions or errors contained in this document. MyBioSource reserves the right to make changes to this datasheet at any time without notice.

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The Thermo Scientific DyNAmo HS SYBR Green qPCR Kit contains a modified Thermus brockianus DNA polymerase and all other reagents required for qPCR. The user only needs to add PCR primers and template DNA. The modified DNA polymerase in this kit incorporates a nonspecific DNA-binding domain that provides physical stability to the DNA-polymerase complex. ROX Passive Reference Dye is also supplied with the kit in a separate tube for instruments that require ROX normalization.

Reflexes

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For research use only. It should not be used in diagnostic procedures.

Specifications

To use with (application): Real-time PCR (qPCR)

For use with (team)

System 7000, System 7300, Rapid System 7500, System 7500, System 7700, Rapid System 7900HT, System 7900HT, BioRad CFX96™, QuantStudio™, ViiA™ System 7, Bio-Rad CFX384 Touch™, Roche LightCycler 480, Detection System for 5700 sequences, StepOnePlus™ System, Eppendorf MasterCycler RealPlex, Corbett RotorGene, StepOne™ System, Stratagene Mx4000, Stratagene Mx3005P, Stratagene Mx3000P, MJ Opticon, MJ Chromo4, Cepheid SmartCycler

Game type: DyNAmo HS SYBR Green qPCR Kit

Product line: Dynamo

Kind of product: SYBR real-time PCR master mix

Enough for: 500 reactions of 20 μL each, 200 reactions of 50 μL each

Concentration: 2X (DyNAmo HS SYBR Green qPCR Mix), 50X (ROX Dye)

Detection method: SYBR

Format: Tube

GC-Rich PCR Yield: High

No. of reactions: reactions

PCR method: qPCR

Polymerase: Tbr DNA polymerase

Reaction speed: Standard

Example type: DNA, dsDNA

Volume: 5x1ml

Content and storage

Contains:

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Store at -20°C

Product Description

AmoyDx® Novel Coronavirus (2019-nCoV) Detection Kit is a rapid and sensitive real-time RT-PCR assay that combines reverse transcription and PCR amplification in a one-step procedure for the qualitative detection of coronavirus SARS-CoV-2 with viral RNA extracted from human respiratory samples. The kit simultaneously detects ORF1ab (open reading frame, ORF1ab) and conserved regions of N (nucleoprotein) of the new coronavirus, with an internal control system to monitor the entire PCR process.

Principles of the Procedure

The kit is based on real-time RT-PCR technology, it combines reverse transcription and PCR amplification in a single-step procedure. It is designed for specific amplification of the conserved regions ORF1ab (open reading frame, ORF1ab) and N (nucleoprotein, N) of the new coronavirus SARS-CoV-2 in the viral RNA. The target region is amplified with various specific primers and detected with fluorescence probes. Internal non-competitive control is included in the nCoV RNA Detection System to assess RNA quality and monitor the entire PCR procedure.

The kit is composed of nCoV Reaction Mix, nCoV Enzyme Mix and nCoV Positive Control.

1) The nCoV reaction mix includes a nCoV RNA detection system and an internal control system. It contains primers and FAM-labeled probe specific for the ORF1ab gene of SARS-CoV-2, ROX-labeled probe specific for the N gene of SARS-CoV-2, and VIC-labeled probe specific for the internal control. The internal control system is designed for a housekeeping gene as a reference gene to assess the quality of the RNA and monitor the precision of the experimental operation, which may lead to false-negative results.

2) The nCoV enzyme mix contains reverse transcriptase for reverse transcription of viral RNA and reference gene RNA into cDNA, Taq DNA polymerase for PCR amplification, and uracil-N-glycosylase to prevent carryover contamination. PCR amplicon.

3) The nCoV positive control contains a nucleic acid template of the SARS-CoV-2 coronavirus ORF1ab and N genes and the housekeeping gene.

Description

Invitrogen dGTP is prepared at a final concentration of 100 mM in highly purified water. 100 mM dGTP is suitable for use in a polymerase chain reaction (PCR), sequencing, stuffing reactions, nick translation, cDNA synthesis, and TdT tracking reactions.

Invitrogen’s dNTP manufacturing meets the highest industry standards:

• Manufactured under ISO 9001
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Features include:

• Chemically synthesized
• pH 7.5
• >99% purity confirmed by HPLC
• Stable for 2 years at –20°C
• Free of qPCR, PCR, reverse transcription inhibitors
• Free of DNases and RNases
• Free of human and E. coli DNA

Specifications

Shipping condition: Approved for shipment on wet or dry ice

Concentration: 100mm

Label or tint: Unlabeled

Quantity: 250 litres

Content and storage

The nucleotide is supplied as a convenient, ready-to-use solution at a concentration of 100 mM in highly purified water (pH 7.5). Store at -20°C.

Applications:

•  qPCR, RT-qPCR
• PCR, RT-PCR, cDNA synthesis
• High-fidelity, long-range PCR
• Isothermal amplification
• DNA marking
• Cloning
• Sanger sequencing and next-generation sequencing (NGS)

For research use only. It should not be used in diagnostic procedures.

Bottom

Peanut sensitization does not necessarily indicate a clinical peanut allergy, and uncertainty about whether or not a true peanut allergy exists can lead to increased anxiety and decreased quality of life for patients and their families. The gold standard for clinical diagnosis of peanut allergy is an oral food challenge, but this method is time-consuming and can cause severe allergic reactions. Therefore, it would be beneficial to develop a tool to predict clinical peanut allergy in peanut-sensitized individuals whose peanut allergy status is unknown to better determine who requires an oral food challenge for diagnosis.

Methods

Two separate studies were conducted. In Study 1, we recruited 100 participants from the McMaster University allergy clinic and community allergy outpatient clinics in the greater Hamilton area. We examined 18 different participant variables and used univariate and multivariate logistic regression analysis to determine how well these variables, alone and in combination, were able to predict clinical peanut allergy status.

In Study 2, we performed a retrospective chart review of a second cohort of 194 participants to investigate the reproducibility of our findings. This was a matched case-control study in which 97 peanut-allergic participants were matched for sex and age with 97 non-allergic control participants.

• Skin prick test measurements

The forearm was prepped with alcohol and peanut extract (ALK-Pharmaceuticals, Mississauga, ON, Canada) was applied to the skin of the dorsal forearm. A sterile metal lancet (HollisterStier, Spokane, WA, USA) was used to pierce the skin below the allergen droplet. Wheal size was measured by skin prick test after 15 min. The tape was placed on the dorsal forearm and the outline of the wheel was traced. Two different study nurses measured the widest diameter of the wheel.

• Plasma measurements of total and peanut IgE

Total IgE was measured with Image 800 (Beckman Coulter, Mississauga, ON, Canada) and peanut-specific IgE antibodies were measured with the Phadia 250 (Thermo Scientific, Waltham, MA, USA).

• Cytokine measurements

Mononuclear cells were isolated from 30 to 40 ml of blood by density gradient centrifugation after lysing red blood cells with AKC lysis buffer. Cells were resuspended in RPMI supplemented with 10% FBS, 1% L-glutamine, 1% penicillin/streptomycin, 55 µM 2-mercaptoethanol (Thermo Scientific), 1 mM sodium pyruvate, 10 mM HEPES, and 0 MEM NEAA. 0.1 mM (Thermo Scientific). Scientific).

Viable cells were counted by Trypan Blue exclusion (Thermo Scientific) and resuspended at 8 × 10 6 cells/mL. 125,000 live cells per well were cultured in triplicate in medium alone or with 50 µg/mL/well crude peanut extract in flat-bottom 96-well plates (BD Biosciences, Mississauga, ON, Canada). After 5 days of culture at 37°C and 5% CO2, triplicates were pooled, centrifuged, and cell-free supernatants were collected and stored at -80°C until further analysis. Cytokines in cell-free supernatants were quantified using Luminex (Millipore Canada Ltd, Etobicoke, ON, Canada) following the manufacturer’s instructions.

• Statistic analysis

Each predictor was entered into a univariate logistic regression analysis to determine if it was associated with the primary outcome: peanut allergy clinical status. We then generated cumulative models composed of multiple predictors using multivariable logistic regression. All univariate and multivariate analyzes included all 69 study participants from Groups 1, 2, and 4.

For all models, parameter estimates were obtained for each predictor and expressed as odds ratios with corresponding 95% confidence intervals and associated p-values. p-values ​​are reported to 4 decimal places.

Hierarchical models were compared to determine if the model with the greater number of predictors was statistically significantly better at predicting the primary outcome than the model with fewer predictors. This was done by comparing the −2 Log-Likelihood statistics of the models. For each model, the area under the receiver operating characteristic (ROC) curve was reported as a measure of discriminability.

The best model was used to predict the peanut allergy status of Group 3 participants and to determine the predicted probability (Pr) that each participant had a clinical peanut allergy. Using Pr, we classify each individual as peanut allergic or not, based on a specific cutoff point. We chose this cutoff point to eliminate false negatives and maximize true positives in the data set.

Results

Peanut skin prick test wheal size was the best predictor of clinical peanut allergy in both study cohorts. For every 1-mm increase in wheel size, the odds ratio that an individual had a clinical peanut allergy was 2.36 in our first cohort and 4.85 in our second cohort. No other variable came close to the predictive power of wheal size.

Conclusions

Peanut skin prick test wheel size is a strong predictor of clinical reactivity to peanuts. The findings of this study may be useful in guiding physicians’ decision-making regarding the diagnosis of peanut allergy.