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  1. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    ...in PubMed and choose the appropriate publication. This publication will be listed at the top of the final... with publication details or permission to distribute, or Addgene finds your journal publication describing...classified in multiple categories, please check all of the appropriate categories (e.g., for a lentiviral... "Published plasmids" or "Unpublished or pre-publication plasmids" and set a desired distribution status...quickly associate your plasmids with the original publication where you described them. Search for your article...plasmids. You can submit "Unpublished or pre-publication plasmids": Unpublished plasmids or plasmids that...accepted. Addgene also encourages submitting pre-publication plasmids so they can be available when the paper...

  2. Isolating Genomic Regions of Interest with the CRISPR System

    Type
    Blog Post
    ...affinity purification with an anti-Cas9 antibody. These different tag systems and affinity purification schemes...genomic alterations. These studies utilize a catalytically inactive form of Cas9 known as dCas9 (Jinek ...interest targeted by a gRNA. CRISPR-mediated purification of a specific genomic region In 2013, the Hodaka...Subsequent mass spectroscopy studies allowed for the identification of proteins associated with the IRF-1 locus...steps (Fujita et al., 2013). CRISPR-mediated purification of a specific genomic region alleviates these...the initial report describing CRISPR-mediated purification of a specific genomic region, numerous adaptations...different fields of study. In addition to the identification of DNA binding proteins, this method can characterize...

  3. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

    Type
    Blog Post
    ...Technical Tips). Minigenome replication and transcription In order to replicate the minigenome, the viral...transcription and replication strategies of Marburg virus and Ebola virus by using artificial replication systems...allowing researchers to study aspects of the EBOV replication cycle under BSL-2 conditions (4). What is a ...that are crucial cis-acting signals for genome replication and viral gene transcription (6). The EBOV genome...5’ ends of the genome which are required for replication and transcription (Figure 1). All viral genes...cis-acting signals necessary to direct minigenome replication and reporter gene transcription by the EBOV polymerase... in 2, 5). The minigenome is capable of being replicated and transcribed in the cell when the appropriate...

  4. Tips from the Repository Trenches: Using Barcodes to Track Samples

    Type
    Blog Post
    .... Tracking samples - it's all about location, location, location There are many different types of samples...but we also have a dedicated and detail-oriented lab team that works carefully to catch mistakes. Each sample...assigning a physical location to your samples, include as much detail about that location as possible. Are...sample a unique location in a specific freezer will help ensure that samples are easy to locate in freezers...also help us keep track of the tubes’ physical location in the lab. These barcodes additionally allow ...Addgene. Each sample at Addgene has four layers of location which can all be extrapolated from the 2D barcode...Plate Freezer Rack Freezer Tubes are assigned a location within a plate that can hold up to 96 tubes (see...

  5. 6 Tips for Grant Writing

    Type
    Blog Post
    ...Series 4. Become a science communicator More and more fellowship applications are asking for a 200-300 ...good quality application. Even following this advice, only about 50% of my grant applications have been ...do need to be able to research and write your application, then give it to someone else to read and / or...it). It’s easy for reviewers to spot when an application is rushed (whether the idea isn’t properly researched...researched / developed or the application is full of typos) and these are unlikely to get past the initial...someone else in the department) to read over your application and give you feedback. They’ve most likely been...alterations before submitting - handing them your application 3 days before the deadline isn’t going to be ...

  6. Antibodies 101: Affinity Tags

    Type
    Blog Post
    ...cleaved after purification, so as not to perturb protein function for downstream applications. Tag cleavage...thoughtful planning, affinity tags can make protein purification a cinch. Types of affinity tags, the proteins... Affinity tags are used primarily as protein purification tools (and can have secondary functions as detection...single protein is expressed, with the intent of purification, it will be expressed along with all other cellular...available antibodies, resins, beads, and other purification aids readily available. Choosing the best tag...tag to remain on your protein for downstream applications Below, we describe a few of the most common...cleavage must be performed after primary purification and requires a second clean up step to remove the cleaved...

  7. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    ...serially transfected using a cationic vehicle. The following synthetic modifications are used to protect the... to verify there’s no genomic integration Replicating EBNA1 episome Non-integrating Medium Medium ...creates heterogeneous iPSC cell lines, which can complicate comparisons made between lines. Incomplete silencing...Retroviruses are not considered safe for clinical applications.     Lentiviral: Lentiviral vectors infect dividing...lentiviral vectors insert in different genetic locations in each transduced cell, so there’s unavoidable...unavoidable heterogeneity between clones. This can complicate comparisons made between samples, i.e. transgene...extent of reprogramming could be affected by the location of insertion. Lentiviral insertion can also disrupt...

  8. Plasmids 101: Broad Host Range Plasmids

    Type
    Blog Post
    ...ori2, ori3) and replication initiation proteins (RepA, RepB) necessary for replication in various hosts...the ori and allow for replication. However, if plasmids have their own replication machinery such as Rep...different hosts that a plasmid can transfer to, replicate in, and persist in. Some plasmids have a narrow... while others have a broad host range and can replicate and stably maintain the genes they carry among.... These features are: The plasmid origin of replication (ori) contains structural elements that are versatile...structure allows plasmids to utilize diverse host replication machinery. For instance, the ori typically contains... iterons that serve as recognition sites for replication initiation proteins. These iterons vary in their...

  9. Summer SciComm: Preprints

    Type
    Blog Post
    ...their CV or grant applications. A number of organizations (such as ASAPbio) advocate for preprints, and...the most important part of your research is communicating the results of the work. Yes, those control ...any impact! There are lots of ways scientists communicate with each other: from formal mechanisms like ...poster or talk, or submitting patent or grant applications; to informal settings like lab meetings, social...of the most significant shifts in scientific communication is the rising use of preprints, or scientific...publishing in a journal.  Accelerating scientific communication Preprints have been common in fields like math...under a CC BY-SA license. Many scientists and advocates have argued that while peer review is valuable...

  10. Site Directed Mutagenesis by PCR

    Type
    Blog Post
    ...desired modification. Finally, the positive clones are sequenced to confirm the desired modification and ...polymerase activity (for amplification), 3’->5’ exonuclease activity (increases amplification fidelity), and no... genome modifications. Occasionally, multimerization of the PCR primers can cause duplication of the primer...the desired modification have been identified (and confirmed to be free of primer duplications), final validation...and the absense of additional modifications.   Site-directed mutagenesis of Plasmids. A) Generation..., which flank the targeted region. Upon PCR amplification, the template is removed by DpnI restriction...extension time to match the size of your template. Amplification of GC-rich plasmids is facilitated by the addition...

  11. Viral Vectors 101: Inverted Terminal Repeats

    Type
    Blog Post
    ... ITRs serve as the origin of replication and are required for replication, packaging, and vector persistence...viral rep (replication) and cap (capsid) proteins, which bind to the ITRs and initiate replication. ITRs are...structures, ITRs are prone to replication errors and disruptive mutations and truncations that can make vector ...binding site for Rep78 and Rep68 to initiate replication and a terminal resolution site that serves as...are noted. Created with biorender.com. ITR modifications and types  Since ITRs are one of the most important...important elements of AAV and rAAV vectors, modifications can affect gene transfer efficiency and transgene...are thought to be self-repaired during the AAV replication process (Chen et al., 2004). Mutations in the...

  12. Viral Vectors 101: What is a Virus (and a Viral Vector)?

    Type
    Blog Post
    ...methods of replication. In fact, an entire classification scheme, known as the Baltimore replication classes...that in a bit! Viral genomes and replication When it comes to replication, viruses tend to march to the ...developed around how they replicate (Figure 2). Figure 2: Baltimore replication classes. Image credit: ...an intermediary DNA replication step between DNA and mRNA. DNA genome replication can involve an RNA or...cell. The cell then uses its own machinery to replicate the viral genome and proteins. This is, for the...before being translated into proteins. RNA viruses replicate their genomes using a variety of RNA or DNA intermediaries... bacteria and eukaryotes, which use a binary replication process whereby one cell copies itself and “splits...

  13. Summer SciComm Series: Master of SciComm

    Type
    Blog Post
    ...begun my journey towards a career in science communication, an established field of inquiry with which ...University of Otago’s Department of Science Communication the following year and, having thoroughly enjoyed...straight on to a master's degree in science communication. The first year of the course involved a whirlwind...world of industry. Along this road, science communication graduates are often well-equipped. Many institutions...truly benefit others. Herein, a good science communicator—with an understanding of how to appeal to a ...pursuit of public engagement. Studying science communication is an equally fruitful endeavor for those embarking...towards academia. Many of those entering science communication (myself included) come from a scientific background...

  14. Plasmids 101: NGS Quality Control for Pooled Libraries

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    Blog Post
    ...would be a good time to talk about the amplification and verification processes we use to ensure high-quality...your own experiments. Library amplification 101 Library amplification seems straightforward: E. coli ...transformed with the library DNA, they grow and replicate, and then their DNA is harvested, much like one...library has its own depositor transformation and amplification protocol, but the overall workflow is similar...ll need to remember when undertaking library amplification and sequencing. Maintain representation of the...electrocompetent Stbl4 cells when performing amplifications, and we use electroporation to ensure a good...your sequencing results represent a successful amplification? To provide a point of reference, Addgene commonly...

  15. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ...potential applications of CRISPR/Cas9 in genome-engineering. One particularly attractive application involves... the perfect voltage indicator and optogenetic actuator pair. Ideally, indicators should possess fast ...random genomic location than to express the tagged protein from a defined genomic location or from its endogenous...temporal control over CRISPR/Cas9 mediated genome modification and gene expression. The plasmids associated... system widely accessible for many potential applications. Polstein LR & Gersbach CA, Nat Chem Biol ...other ions In BRET, A mutant Renilla luciferase, catalyzes the oxidation of its substrate, luciferin, which...amenable to both high and low throughput screening applications. The system can be readily modified to use different...

  16. Plasmids 101: Restriction Cloning

    Type
    Blog Post
    ...them. An easy way to do this is gel purification. In gel purification, you use a voltage difference across...backbone. Sample results indicative of successful and unsuccessful ligations are indicated below. A successful...grow overnight cultures for DNA purification. The simplest purification you can do is a miniprep, but if...digests. Of course there’s much more detail and verification required for the process to work well, so let...backbone. Because you lose some DNA during the gel purification step, it is important to digest plenty of starting...prior to the ligation step or prior to the gel purification step, depending on the phosphatase you choose...   2. Isolate Your Insert and Vector by Gel Purification     Now that you’ve cut your insert and vector...

  17. Plasmids 101: Shuttle Vectors

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    Blog Post
    ...are used across species: replication, selection, and expression. Replication It’s usually not enough to...chromosome has a single origin of replication (ori), where DNA replication begins, so a bacterial plasmid...Eukaryotes are a bit more complicated. Yeast vectors may contain an autonomously replicating sequence (ARS), a...fast-growing populations of cells, the vector needs to replicate alongside its host, or it will be lost from the...compatible ori that will be recognized by its host’s replication machinery. A shuttle vector designed for unrelated...experiment. Yeast shuttle vectors have these replication sequences for their yeast targets as well as ...episomal vectors is to include viral oris to drive amplification, which helps increase cellular uptake, but this...

  18. Viral Vectors 101: Preparing Pooled Libraries

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    Blog Post
    ...you.  Starting from plasmids requires amplification, verification, and lentiviral production steps before...the same proportion after amplification as they were prior to amplification. And as anyone who has worked...process for using pooled lentiviral libraries. A) Amplification, validation, and lentiviral generation of a ...pooled library is transformed into E. coli for amplification. Transformed E. coli are allowed to grow on ...into HEK cells allows production of lentivirus. Purification of this lentivirus results in the pooled library...//pubmed.ncbi.nlm.nih.gov/27366891/   Amplification When you request a DNA pooled library from Addgene...might receive a similarly-sized aliquot from an amplification done by Addgene.) This small aliquot is not ...

  19. Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

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    Blog Post
    ...activity of CcdB. Applications of ccdB in plasmid cloning Perhaps the most widely used application of CcdB is...selection markers. Restriction modification systems Because restriction modification systems consist of two enzymes...used as biological tools for a wide range of applications in molecular biology or in vivo studies. Let...types of plasmid addiction systems and their applications. The discovery of toxin-antitoxin systems Toxin-antitoxin...CcdA (antitoxin) where CcdB inferes with DNA replication by binding the GyrA subunit of DNA gyrase and...construct. Other toxin-antitoxin systems and their applications MazE/MazF Aside from CcdB/CcdA, there are many...counterselectable marker to create unmarked genetic modifications in the yeast Pichia pastoris. By combining both...

  20. Plasmids 101: Control Plasmids

    Type
    Blog Post
    ...two types of replicate controls: technical and biological. In general, technical replicates can be thought... employ transfection, negative, positive, and replicate controls.  Transfection controls: empty vector... Plasmid B (as internal control) This result indicates that the transfection was not successful due to...the interrogation of the independent variable. Replicate controls: technical and biological Reliable results...derived from one source. Conversely, biological replicates ARE independent and can be thought of as "reproducibility...reproducibility controls". Biological replicates are what makes up your sample size (aka your n value)"... provide more in depth information. Technical replicates An example of a technical control is the transfection...

  21. Plasmids 101: Using Transposons in the Lab

    Type
    Blog Post
    ... It generally stays put, which makes it easy to locate in a genome. But there is a type of mobile DNA,... hyperactive and likes to jump around from one location in the genome to another. This jumping is what...most transposable elements (TEs) results in the duplication of this target site sequence at the site of insertion...This is similar to how retroviruses, like HIV, replicate. Class I TEs do not encode a transposase enzyme...enzyme.  Class I TEs are considered replicative since every time they jump they make a copy of themselves...retroviruses both in their structure and mechanism of replication. They contain two genes: gag and pol. The pol...they move. Most Class II transposons have a non-replicative “cut and paste” mechanism of transposition: they...

  22. Important Considerations When Using AAVs

    Type
    Blog Post
    ...Student at the University of Manitoba. Location, Location, Location! Failure to Plan (for Storage) Is Planning...Viral Vector Blog Posts from Addgene 1. Location, location, location! What do buying real estate and using...for a house and choosing an AAV are… location, location, location! Just like this common real estate mantra...important factors related to location. Location (1): What tissue? Location (2): Where in the tissue/which...which cell type? Location (3): Traveling from the original location - Axonal Transport in the CNS What...particular experiment! Traveling from the Original Location - Axonal Transport It has been demonstrated that...previous literature using AAVs for DREADD delivery indicates ~7-21 days are required for the onset of gene ...

  23. CRISPR 101: Cytosine and Adenine Base Editors

    Type
    Blog Post
    ...editors catalyze purine-to-purine or pyrimidine-to-pyrimidine base changes. Solid red arrows indicate a conversion...conversion directly catalyzed by the base editor. Dotted red arrows indicate the change introduced to ... point modifications. CRISPR base editing persists through multiple cell divisions, indicating that this...target modification in primary T cells, making them a promising tool for cell therapy applications. Dual...Base editing publication highlights In the table below, you'll find a list of publications we've highlighted...Early CRISPR applications were often limited by the low editing efficiency of homology-directed repair...following base editing and DNA repair. Black arrows indicate natural cellular processes. BER = base excision...

  24. Adenoviral Vector Production and Troubleshooting

    Type
    Blog Post
    ...Each round of amplification should result in a 10-100-fold increase in virus. Purification (2 days) - Purification... Purification is required if rAdV is to be used in vivo. The standard method for purification of rAdV ...vectors (AdV) are attractive vectors for research applications and gene therapy: they can be produced at high...AdVs is what makes them ideal candidates for applications in oncolysis and vaccination!) In the 65 years...Adenovirus-mediated gene delivery: Potential applications for gene and cell-based therapies in the new...genes E1 and E3. Without these genes, AdVs cannot replicate on their own but can be produced in E1-containing...The ever popular pAdEasy system belongs to this category. 3rd Gen AdV (Gutless or high capacity AdV) are...

  25. Anatomy of a Plasmid Page at Addgene

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    Blog Post
    ...lab while the publication page contains all the plasmids deposited from the same publication. Next up, we...number given to every plasmid in our catalog - if you know the catalog number of the plasmid you are searching... Addgene’s plasmids is an indication of popularity. A yellow flame indicates that a plasmid has been ordered...through the depositing lab page, then onto the publication page, and finally on down to the individual plasmid...deposit process and these can be found in this location as well. These maps make it easy for scientists...Great! Now, if I want to use this plasmid in a publication, how do I know who to acknowledge? Depositing...plasmids that are available through Addgene. Publication: If the plasmid has been published, the paper...

  26. What’s in Your Tube?: A Quick Guide to Measuring DNA by Spectrophotometry

    Type
    Blog Post
    ...present.  Purification reagent contaminants The reagents used during nucleic acid purification can leave...Selecting Your Plasmid Purification Kit Resources on Addgene.org DNA Quantification Protocol Recovering... and are ready to move on to your downstream application — but wait! Do you know how much plasmid DNA ...(Figure 1B). The A260/230 ratio can be a good indicator of protein contamination, but a number of other...researchers traditionally use the A260/280 ratio as an indicator of protein contamination. However, this ratio ...230 ratio of a sample. Ethanol carry-over from purification protocols is also a common concern, and while... guanidine thiocyanate (used in many plasmid purification kits), which can dramatically reduce the A260...

  27. Great Results Start with Great Standard Curves

    Type
    Blog Post
    ... be run in duplicate or triplicate. The closeness of the data points generated by replicate values provides... the standard dilution series in duplicate or triplicate. Replicates allows you to calculate the curve... deviation of each replicate measurement divided by the mean of all the replicates multiplied by 100. ...regularly, and mix samples very well.  Use duplicates or triplicates to increase the accuracy of your experiment... critical for a number of popular scientific applications, and the quality of a standard curve can make...diluted standard as a function of concentration on a scatter plot. For the BCA example, the concentration of...correlation between the actual values and the predicate values and measures how well the data points fit...

  28. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

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    Blog Post
    ...of its biggest limitations is that sequence modifications can only be made at restriction enzyme cut sites...homologous recombination. It allows for direct modification of DNA within E. coli and is independent of ...engineering.  It can be used to make an assortment of modifications: insertion and deletion of selectable and non-selectable...expressing the lambda red enzymes. These enzymes then catalyze the homologous recombination of the substrate ...however, only Beta is required when generating a modification with an ssDNA substrate.  Gam: Gam prevents ...regions flanking the target site).  The best applications for dsDNA inserts include: large insertions ...nucleotides long with the desired alteration(s) located in the center of the sequence. Since lambda red...

  29. Antibodies 101: Selecting the Right Antibody

    Type
    Blog Post
    ...your specific application. Consider also that there may be variations within an application. Take immunohistochemistry...website highlights a variation of your application, a publication successfully used it in a similar way...choosing an appropriate antibody first look at the application-specific data provided on the manufacturer’s ... a linear antigen tend to work very well for applications that denature a protein to its primary (linear...the most similar to yours. Take a look at the publications cited on the manufacturer’s website, which may...plan to. To secondary or not to secondary?  The application will also determine whether you should use an...antibody. If you are trying to visualize the precise location of a protein in a tissue section then a directly-conjugated...

  30. Plasmids 101: Cre-lox

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    Blog Post
    ...recombinase catalyzes the site specific recombination event between two loxP sites, which can be located either...maintained).  Translocation: If the sites are on separate DNA molecules, a translocation event is generated...important plasmid elements – promoters, origins of replication, protein tags, and antibiotic resistance markers...creating (excuse the pun) specific, targeted DNA modifications in transgenic animals, embryonic stem cells,...proteins or co-factors, thus allowing for broad applications in a variety of experiments. LoxP (locus of ...and the canonical loxP sequence is ATAACTTCGTATA-GCATACAT-TATACGAAGTTAT. The loxP sequence does not occur...Therefore, inserting loxP sites at deliberate locations in a DNA sequence allows for very specific manipulations...

  31. Typing CRISPR Systems

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    Blog Post
    ... your own research! Classification Structure Principles of CRISPR classification While CRISPR systems...Shakespeare for the first time. The discovery and application of CRISPR-Cas9 kicked off over a decade of frenzied...properties. This led to a need to develop a classification system for the array of CRISPR technologies...technologies. Learning the general principles of this classification system will help you gain a deeper appreciation...now seem neatly organized into their respective categories, arriving at a consensus was not as straightforward...multi-pronged analysis to sort effectively. The current classification system relies on a combination of sequence ...a similar function. After analysis, CRISPR classifications were placed into a fairly simple hierarchy:...

  32. Common Injection Routes in Mice

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    Blog Post
    .... It sounds complicated but has a pretty low learning curve! Restraint: scruffing Location: throat Gauge... Restraint: mechanical restraint or anesthesia Location: tail vein or retro-orbitally Gauge: 27–30 Volume...practice to ensure the proper injection site has been located and to avoid potential off-target pokes to nearby...nearby organs. Restraint: scruffing Location: peritoneal cavity Gauge: 25–27 Volume: < 2–3 ml Skill required...getting a vaccine. Restraint: mechanical restraint Location: thigh Gauge: 25–27 Volume: < 0.02–0.05 ml Skill...Restraint: back scruffing or mechanical restraint Location: back or hind legs Gauge: 25–27 Volume: < 2–3 ...area must be shaved first. Restraint: anesthesia Location: between skin layers Gauge: 26 Volume: < 0.05 ...

  33. Tips for a 1st time AAV user (by a Rookie AAV user)

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    Blog Post
    ...which provides the AAV replication (rep) and capsid (cap) genes. AAV replication uses the host’s polymerase...this AAV purification video! It didn’t exist when I did my first iodixanol gradient purification, but I ...In this post, I’ll share some AAV production, purification, and titration tips, while also summarizing ...adenovirus genes which help mediate AAV viral replication; and 3) the transfer plasmid which encodes a ... that aids in the homologous recombination of duplicated regions like the ITRs. Restriction digest screening...consider performing PEG precipitation. Instead of sonicating the cell pellet, I lyse the cells with this AAV... with benzonase immediately before iodixanol purification. This approach gives me a bit of flexibility...

  34. Antibodies 101: Epitope Tags

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    Blog Post
    ...(stay tuned for our affinity purification tag blog!).   Applications of epitope tags   The...success rates across all applications. You can find FLAG antibodies on Addgene’s catalog including a mouse monoclonal...protein purification is not ideal with Myc, as low pH conditions are required during the purification, which...experienced. Finding an antibody that works for your application, specifically detects your protein, is species...and have been widely used across species and applications. In this blog, we will review the most common...that are bound by antibodies for the purpose of purification or detection of the tagged protein. These peptides...not exist, or they don’t exist for a specific application, or they exist but are unreliable for a variety...

  35. Plasmid Cloning by PCR

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    Blog Post
    ...not cut within your insert Are in the desired location in your recipient plasmid (usually in the Multiple...amplify and design primers that will bind to and replicate it. The following image shows the ends of the ...Forward Primer will use the sequence 5'-ATGTGGCATATCTCGAAGTAC-3' for the region that binds the ORF and...primer, making our Forward Primer 5'-GAATTCATGTGGCATATCTCGAAGTAC-3'. Many restriction enzymes do not cut...final Forward Primer sequence of 5'-TAAGCAGAATTCATGTGGCATATCTCGAAGTAC-3'. For the Reverse Primer, the design...need to use the reverse complement to get PCR amplification. We can start similarly, taking the final 18...of the ORF, including the stop codon (5'-TGGCATATCTCGAAGTACTGA-3'), then adding NotI (GCGGCCGC) and then...

  36. Simplify Cloning with in vivo Assembly

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    Blog Post
    ...3’ end binds template DNA for PCR amplification, while modifications and homologous sequences are encoded...multiple modifications A key feature of IVA cloning is the ability to combine multiple modifications. Any ...’ Check your amplification by gel electrophoresis. In our experience, good amplification after PCR means...gain traction? It is likely that both vector amplification with low fidelity polymerases and the cost of...this approach requires no special reagents or purification kits, it is low cost and can be immediately ...to produce the linear fragments, as desired modifications and homologous regions can be encoded in the...are perfectly fine, yet performing multiple modifications will require ultracompetent (109 CFU/μg) cells...

  37. CRISPR-based Genome Editing Systems For Editing in E. coli

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    Blog Post
    ...far, we’ve used the MUCICAT system in two ways: In our initial paper, we used MUCICAT to optimize the gene...industrial strain. Not only is MUCICAT better, it is faster. Using MUCICAT only needs 2 newly constructed...integration using CRISPR-associated transposases” or MUCICAT. Here, we’ll describe each of these CRISPR systems...providing users with a complete editing process. The MUCICAT system While the above systems are great for experiments...chromosomal integration (ex: genome editing), our MUCICAT system which is based on the CRISPR-associated ...unstable and lack precise control over copy number. MUCICAT system contains 4 plasmids: pDonor, pTnsABC, pQCascade... crRNA can be customized as well, including concatenations into crRNA arrays targeting multiple different...

  38. Visualizing Protein Turnover In Situ

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    Blog Post
    ...This application of AHA labeling is known as fluorescence noncanonical amino acid tagging (FUNCAT, Dieterich...experiments. Visualizing newly synthesized proteins with FUNCAT Professor Schuman’s technique applies metabolic...the whole de novo synthesized proteome. However, FUNCAT alone cannot be used to determine the turnover ...specific protein. Professor Schuman’s lab showed that FUNCAT’s usefulness can be extended to determine the turnover...ligation assay (PLA). The clever combination of FUNCAT and PLA PLA was originally published by the Söderberg...DNA strands take part in rolling circle DNA amplification. The amplified DNA segment is labeled with fluorescent...fluorescent probes that enable visualization of the location of the protein-protein interaction. To study protein...

  39. Summer SciComm Series: Cognitive Load

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    Blog Post
    ...from other sources. Mode of communication Different modes of communication take up different amounts of...your audience needs to understand what you’re communicating. While I’m sure that there are loads of ways...new languages!) Jargon has a place in science communication, but the more you use, the higher cognitive ...This can be obvious, in that a lot of science communication is helping people understand new concepts. You...intuitive, or logical to a layperson. When you’re communicating ideas that you think are clearly connected, ...pick and choose what concepts and details to communicate. It’s okay (and encouraged) to leave blank spaces...over some aspects when needed. The trick is to communicate your key points well enough that the audience...

  40. Plasmids 101: What is a plasmid?

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    Blog Post
    ...Description Origin of Replication (ORI) DNA sequence which allows initiation of replication within a plasmid...point for PCR amplification or sequencing. Primers can be exploited for sequence verification of plasmids...important. Our “Plasmids 101” series designed to educate all levels of scientists and plasmid lovers - serves...plasmids are small circular pieces of DNA that replicate independently from the host's chromosomal DNA....virulence. All natural plasmids contain an origin of replication (which controls the host range and copy number...Minimally, lab-created plasmids have an origin of replication, selection marker, and cloning site. The ease... plasmids and the ability of plasmids to self-replicate within a cell make them attractive tools for the...

  41. Plasmids 101: Protein Expression

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    Blog Post
    ...of complex post-translational modifications. Extraction and purification of recombinant proteins from ...modified via an array of post-translational modifications including protein folding, formation of disulfide...mRNA and the addition of post-translational modifications Learn more molecular biology in our Plasmids... experimental, biotechnological, and medical applications. Researches can visualize proteins in vivo by...expression systems used for protein production and purification. These include mammalian, insect, bacterial,...separated from contaminants using a variety of purification methods. Deciding which expression system to...protein you need   Your plans for downstream applications In this blog post we will summarize some of...

  42. A Control for All Seasons

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    Blog Post
    ...results.  Replicates Replicates are another critical control in biological experiments. Replicates can be...of biological replicates such as multiple mice in each group and C, technical replicates or repeated measurements...secondary antibodies.   Application-specific controls Some antibody-based applications have very specific ...then moving on to controls for antibody-related applications. After reading, you will have a better understanding...blot and look for the molecular weight shifts indicative of tau phosphorylation. By including the positive...statistical power of an experiment. Technical replicates are repeated measurements of the same sample ...difficult to obtain meaningful results.  Biological replicates are distinct samples that are treated identically...

  43. SciComm with the Experts at Science in the News Part 1

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    Blog Post
    ...Science Communication Conversation in Science Communication The Purpose of Science Communication #1 Mistake...supporters of science communication. To help you think about ways to effectively communicate your science, we...News at Harvard University. Effective science communication is required to promote public support for research... organization that works to promote science communication. Science in the News hosts a podcast, seminars...That’s right, #ActualLivingScientists, eager to communicate their science. In this first half of our two-...programs SITN runs and learn how they think about communicating science. Listen to the interview with Science...Mistake Scientists Make when Communicating with Non-Scientists How to Approach Different Audiences Listen...

  44. Introducing Addgene's New Search - Find Plasmids More Easily For Your Research!

    Type
    Blog Post
    ... information you are looking for in our catalog or educational resources: plasmid, pooled library, blog...articles. From paper to plasmid Authors regularly indicate that their plasmids are available from Addgene...papers. Maybe they started their deposit prior to publication and were able to include specific plasmid ID ...terms we can use to find materials from this publication. Searching for the title of a paper works You...filters Not every plasmid search will start from a publication. You may have an idea for a plasmid that would...try different terms you may find the available categories and filters change as we now only show options... a second filter, this one from the ‘Requests’ category, will leave us with a single plasmid to consider...

  45. A Career in Grants: How To Become a Grant Professional

    Type
    Blog Post
    ...development, certification, degrees, foundations, coaching and even Twitter Chats dedicated to the Grant...the candidate application process, the candidate’s ability to write a grant application through a written... much more than writing. In fact, many grant applications and grant makers have moved away from long written...Now they require grant seekers to use online applications with significant character limits for the written...written portion of the grant application. They have increased their demand for stronger budgets with clear...duties can range from coordinating the grant application and submitting it, to evaluating the capacity...asked to serve on review panels and to review education materials for publishers. When the community orchestra...

  46. Unique and persistent IDs for improved reproducibility: Addgene now supports RRIDs and compact identifiers for all plasmids

    Type
    Blog Post
    ...about which reagents were used? Unambiguous identification of a reagent is crucial for reproducibility...can lead to wasted time or retractions. Using a catalog number for identifying a reagent can be helpful...reagent online. Furthermore, if the link between the catalog number and reagent description is lost (e.g. a ... supplier is bought out by another company and catalog number changes) the original identifier loses its... but this approach has been adapted for the identification of physical materials that we use in our labs... which serve as unique PIDs within the Addgene catalog. These IDs allow you to unmistakably identify a...item. Given a specific Addgene ID # listed in a publication, you can quickly pull up the associated plasmid...

  47. Live and Let Dye: Self-Labeling Protein Tags

    Type
    Blog Post
    ...Few Complicated Non-natural amino acids 1 Various Many Complicated   Applications Organic...new green fluorescent protein, some affinity purification tags, another version with that same red fluorescent... an enzyme that has been mutated to block its catalytic activity and instead become trapped in a covalent...having to clone a whole new construct for every application. Plus, the photophysical properties of chemical...advantages over fluorescent proteins, opening up new applications.   Figure 1: Live-cell labeling strategies...Although these multi-component systems can be complicated to implement, they can be highly specific with...self-labeling tag and a fluorescent protein) enables applications like multi-color imaging (Figure 2). If you’...

  48. Plasmids 101: Yeast Vectors

    Type
    Blog Post
    ... post on mammalian vectors, will catch you up on the core replication and resistance features of yeast...recombination. Yeast Replicating plasmids (YRp):  These vectors contain an Autonomously Replicating Sequence (ARS...vectors must contain a yeast-specific origin of replication (ORI) and a means of selection in yeast cells...antibiotic selection markers.  The yeast origin of replication (Please note: This first section primarily pertains...ve also noted some features required for the replication of fission yeast, Schizosaccharomyces pombe, ...within a yeast vector determine how the plasmid is replicated and maintained within the yeast cell. These elements...integrated into the host DNA or is independently replicated as an episome. The four main types of yeast plasmids...

  49. Antibodies 101: Introduction to Gating in Flow Cytometry

    Type
    Blog Post
    ... you with two measurements: forward scatter (FSC) and side scatter (SSC). To acquire these measurements...exclude all duplicate events (two cells stuck together) from your single cells, as duplicates can’t be reliably...types of scattering that occur when a cell is pulsed by the laser. Within each type of scattering, you also...to keep it short though, promise! FSC and SSC: scattering To digitally extract your cells based on their...passing cell with a laser and detects the light scattered by that cell at a low angle (FSC) or large angle...: The laser pulse illuminates the cell and is scattered forward at a low angle (FSC) or to the side at...passes through the laser beam and causes it to scatter (FSC/SSC), the detected photons can be represented...

  50. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    ...sensors One of the latest additions to Addgene’s catalog is a new class of sensors for neuropeptides and...doi: 10.1038/s41592-022-01597-x. New glutamate indicator iGluSnFR3 Improving on the glutamate sensor iGluSnFR...They generated variants optimized for different applications (v82 has higher affinity for glutamate but slower...PDGFR.codonopt    Aggarwal A, et al. Glutamate indicators with improved activation kinetics and localization...have developed a new genetically-encoded voltage indicator (GEVI) called JEDI-2P (Liu et al. 2022) optimized...Sustained deep-tissue voltage recording using a fast indicator evolved for two-photon microscopy. Cell. 2022 ...has generated a new genetically encoded calcium indicator using small monomeric NIR fluorescent proteins...
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