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Showing: 201 - 240 of 974 results

  1. Twenty Years of Addgene Sharing: CRISPR

    Type
    Blog Post
    ... the cumulative number of labs who have deposited CRISPR plasmids (Figure 1C), and the cumulative number...distribution and deposits. (A) Cumulative CRISPR plasmids shipped by year. (B) Cumulative CRISPR plasmids deposited...(C) Cumulative number of laboratories that have deposited CRISPR plasmids by year. (D) Cumulative CRISPR... a classic double-stranded break, while modulators regulate transcription and chimeric efforts make a ...Distributing and depositing In Figure 1, you can see cumulative numbers of CRISPR plasmid distribution and deposits...nuclease into three functional groups: nucleases, modulators, and chimeric effectors. Nucleases generate a...distribution and deposits per year, rather than a cumulative total.    Figure 2: Addgene CRISPR deposit...

  2. Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity

    Type
    Blog Post
    ...transformation plate can be manipulated to result in appropriate levels of colony plating densities. Ligase...Outgrowth plated) Correct assemblies per plate Fidelity of assembly (percent correct) Calculated colony ... can plate larger volumes of the outgrowth containing the transformants onto the selection plate. Figure...of assemblies per plate using outgrowth volumes described in Figure 3, with calculated yields from entire...phenotype upon growth on LB/Cam/X-gal/IPTG agar plates, indicating successful reconstruction of the coding...assemblies was achieved by the sequencing of plasmids isolated from blue or white colonies. Sequencing of blue...Figure 2 shows representative transformation plates obtained from 1-, 12- and 24-fragment assemblies of ...

  3. Great Results Start with Great Standard Curves

    Type
    Blog Post
    ...are sigmoidal. Once the trendline is calculated, you can extrapolate the concentration of the unknown sample...software to calculate the data’s coefficient of determination, R2. R2 is the square of the correlation between...could be because there is no correlation or it could mean that the relationship is non-linear and you should...versus the response and use that information to extrapolate the concentration of an unknown sample. For example...the accuracy of the curve, which we will discuss later.       Standard curves must be included every time... the experiment and use the standard curve to calculate its experimental value. Though it varies from ...measurements and the trendline y = 0.6561x + 0.019 calculated. The concentration of an unknown sample is determined...

  4. Droplet Digital PCR for AAV Quantitation

    Type
    Blog Post
    ...used to calculate the concentration of the sample. This concentration can then be used to calculate the viral...generator to the PCR plate. Maintaining a high droplet count is important for calculating the concentration...Addgene, we typically load 3 dilutions onto our ddPCR plate. As the titer is unknown, each dilution should fall...making the dilutions, they are transferred to a new plate containing the mastermix which includes primers ...a droplet reader extracts the droplets from the plate and measures the fluorescence amplitude of each ...positive (blue) and negative (gray) droplets. The no template control wells should have very few positive droplets...approximately 1 positive copy per microliter in the no template control wells. You’ll also notice a reduction ...

  5. Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

    Type
    Blog Post
    ...antitoxin; Modulation of killing (mok), required for hok translation. Here, Mok and Hok translation is coupled...that is regulated by antisense RNA, which base pairs to cognate mRNA to inhibit its translation. The hok...coupled. Translation from the Shine-Dalgarno (SD) sequence of mok results in translation of Mok and Hok...restriction-modification systems onto plasmids allowed cell populations to retain more of these plasmids as compared ...the cell grows resulting in the appearance of unmethylated sites in the chromosome. The more stable endonuclease...chromosome leading to cell death. Antisense RNA-regulated systems Toxin-antitoxin systems are not just restricted...hok/sok system is the first antisense RNA-regulated toxin-antitoxin system discovered. Unlike the systems...

  6. Transferable Skills Guide: External Collaborations

    Type
    Blog Post
    ...we shared materials and equipment between labs. Later I worked with people outside the department to accomplish... connect the imaging community and to create a platform for discussions and potential collaborations. ...loads of opportunities to create communities, platforms, and to be a part of projects that connect people...realize. Being at a university provides the perfect platform for practicing initiative and for training in ...conference calls and make sure to maintain these relationships by keeping in touch by email and social media...media (see the section on “Maintaining Relationships” below). Expand: Collaboration opportunities outside... fun. I just had to remember to approach the relationship with the perspective of my potential collaborators...

  7. Finding nucleic acids with SHERLOCK and DETECTR

    Type
    Blog Post
    ...detected on commercial lateral flow strips, similar to pregnancy tests. With lateral flow strips, the presence...recently adapted the DETECTR platform to detect SARS-CoV-2 using a lateral flow strip format. They published... to develop simple, portable, and inexpensive platforms to reliably detect nucleic acids at the atomolar... to cleave ssRNA or mRNA. It also exhibits a “collateral effect” of non-specific ssRNA cleavage. How does... for nucleic acid detection almost anywhere as lateral flow strips are easy to transport and work rapidly...reliably distinguish between Zika and a closely-related virus, Dengue from multiple sample sources. SHERLOCK...from cell-free DNA fragments as well as health-related single nucleotide polymorphisms (SNPs) from human...

  8. Antibodies 101: The Four ELISAs and When to Use Them

    Type
    Blog Post
    ...antigen present in the sample and is calculated by extrapolating from the standard curve. Because the ...antigen present in the sample and is calculated by extrapolating from the standard curve. Scientists often...same overall process. First users coat a multiwell plate with a capture reagent. The capture reagent may ...of the sample to that of a standard curve to extrapolate the concentration of antigen in the sample. A...substrates since they can be read on a standard plate reader as opposed to chemifluorescent, and chemiluminescent...antibody binds to antigen coated on the multiwell plate. In an indirect ELISA a primary antibody first binds...primary antibody. In a sandwich ELISA the multiwell plate is first coated with a capture antibody that binds...

  9. Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

    Type
    Blog Post
    ...undergo significant post-translational modifications including glycosylation, disulfide bond formation...proteins functionally relative to the untagged protein of interest. To avoid the accumulation of dark fluorescent...is, unfortunately, some investigators realize too late that they have missed the impact of dark, non-fluorescent... fold? Misfolded FPs do not fluoresce. A dark population is not readily apparent and can confound quantitative...construct [1,2], i.e. positioning of the FP sequence relative to cell compartment targeting sequences, which...1) [4,6–8]. Additionally, FPs that encode N-glycosylation consensus sequences are modified with the addition...addition of N-glycans [9]. Both of these posttranslational modifications can disrupt FP folding rendering...

  10. Plasmids 101: Inducible Promoters

    Type
    Blog Post
    ...How are inducible promoters regulated? Inducible promoters can be regulated by positive or negative control...Addgene! Chemically inducible promoters Chemically regulated promoters are among the most common inducible ...protein purification. When arabinose is absent, regulatory protein AraC binds O and I1 sites upstream of...complexed with cAMP activator protein (CAP) can also stimulate AraC binding to I1 and I2 sites. Supplementing...include positive inducible alcohol and steroid regulated promoters commonly used in plant research. ...Example Activator Addgene Plasmid/Kit Alcohol-regulated AlcA promoter AlcR pGGA008 (AlcA) and pGGC011...AlcR) from red flame GreenGate kit Steroid-regulated LexA promoter XVE (synthetic) pFZ19 (yellow flame...

  11. Five Popular Model Organisms

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    Blog Post
    ...externally. Fruit flies are also relatively straightforward to manipulate genetically. Scientists can make...make it easy to study the effects of genetic manipulations. In this blog post, we’ll cover five popular... model organism for scientists as they have a relatively short generation time for mammals - the time ...study diseases such as cancer since mice better recapitulate the complex interactions between cancer cells...are genetically tractable. Mice can be easily manipulated with tools like CRISPR to make transgenic lines...entirely sequenced and is very amenable to genetic manipulation. Yeast cells are great model organism not only...studied in other model organisms that better recapitulate in vivo conditions similar to humans. For example...

  12. CRISPR 101: Targeting Non-Coding RNAs with CRISPR/Cas9

    Type
    Blog Post
    ...enhance or upregulate ncRNA expression. This approach is useful for lncRNAs because they regulate nearby ...They play important roles in many cellular and regulatory processes, but much of their functionality is...unveiled as they are more difficult to functionally manipulate and study in comparison to protein-coding genes...to systems like CRISPR/Cas9 that allow them to manipulate full genes and observe the effects. Luckily for...of disrupting adjacent genes, especially those regulated by or overlapping with the lncRNA (Basak and Nithin...effects can be minimized by disrupting lncRNA regulatory elements that are crucial for transcription and...and Bester 2021). For example, inserting a polyadenylation signal downstream of the transcription start...

  13. No Llamas Required - Synthetic Nanobodies Against Membrane Proteins

    Type
    Blog Post
    ...antibodies bound to an ELISA plate to capture Myc-tagged sybodies. Then, the biotinylated target protein is added...coronaviruses into a llama over several days and isolated special camelid antibodies that bind to the spike...secondary nanobodies blog post.  A rapid selection platform for generating sybodies against membrane proteins...weeks (Zimmermann et al., 2020). This selection platform was designed so that any standard lab can quickly...in the form of mRNA for academic research. The template plasmids for generating your own libraries are...Generation Toolbox! Starting with libraries and a biotinylated (for purification purposes) protein target of...involves using a commercial kit for in-vitro translation of the mRNA libraries. Each mRNA molecule in ...

  14. Transgenic Organisms, Cas9 Gene Drives, and Appropriate Safeguards

    Type
    Blog Post
    ...gene drives” capable of spreading through wild populations. Whereas most genomic changes impose a fitness...could eventually spread through the entire wild population with unpredictable ecological effects. Because...chromosome and its own gene is used as a repair template, thereby converting the heterozygote into a homozygote...building gene drives that can spread through populations of transgenic laboratory organisms but not wild...sgRNA-only drive will spread exclusively through populations that already express Cas9 from an unlinked locus...sequence will only spread in transgenic laboratory populations with that sequence2. Both methods are easy to...performed in regions that don't have resident populations of the relevant mosquito species. Reproductive...

  15. An Introduction to Adenovirus

    Type
    Blog Post
    ...expression, and protein translation. Its genome consists of “early” (E) and “late” genes, and the transition...transition between early and late infection (and therefore between early and late gene transcription) is demarcated...unusual cases of acute hepatitis in the pediatric population. Once ~400 cases had amassed in places around...exist. The life cycle of AdVs is dependent on modulation of the host cell’s cell cycle; inhibition of ...recombinant sequence (transgene) instead.    Although the late genes are usually present in the transfer plasmid...expressed, since the early genes are absent, and the late genes (and their structural proteins, which would... some of the wild-type viruses can persist as a latent infection in certain tissue reservoirs (Pereira...

  16. FLEx Technology and Optogenetics: Flipping the switch on gene expression with high spatial and temporal resolution

    Type
    Blog Post
    ...optogenetic stimulations, they simultaneously recorded the electrical impulses of discrete subpopulations of this... or reward-related behaviors. The high spatio-temporal precision and reversible modulation of FLEx vectors...combined optical and genetic methods to photo-stimulate mammalian neurons and alter synaptic events: a..., 2005; Deisseroth et al., 2006). Despite its relatively recent development, the history of optogenetics...rhodopsins and neurons have in common? Neuronal stimulations are triggered by the movement of ions across...Zhang, 2012; Towne et al., 2016). Optogenetic neuromodulation is made possible by the light: when hit with...viral-infected neurons are subsequently photo-stimulated through fiberoptic cannulas directly implanted...

  17. Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More

    Type
    Blog Post
    ...is its amenability to genetic manipulation. Tools for gene manipulation together with information about...drive biology is to examine the consequences of manipulating genes. One reason for the success of zebrafish...groundwork for understanding the gene networks that regulate processes from development to metabolism to fertility...back to) is the one-eyed pinhead mutant, which regulates a developmental pathway implicated in congenital...morpholinos complementary to a target mRNA inhibit its translation either by blocking initiation or splicing. (RNAi...(RNAi, in contrast, engages existing gene regulatory mechanisms to block activity and does not work easily...compensation, a previously unknown mechanism of gene regulation (El-Brolosy et al., 2019). Generating zebrafish...

  18. Networking Follow-up: I Met a Person. Now What?

    Type
    Blog Post
    ... build the relationship in a non-awkward way?  7 ways to strengthen your new relationships Here are some...Relationships need nurturing. Perhaps you’ve met someone interesting at an event. Or, perhaps you have...contacts you have; it’s about building genuine relationships with people you care about knowing. How do you...some ways to go about strengthening a new relationship with authenticity and not discomfort: Invite the...their new publications via Pubmed, so you can congratulate them when there’s good news.  Send an email ...meeting (in person or virtually) will make the relationship stronger.  If appropriate, invite them to speak...department. Even if it doesn’t work out, it’s flattering to ask.  Show appreciation. Send a thank you ...

  19. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    ...customized to control transgene expression. Gene regulatory elements [promoters and enhancers, miRNA target...IV injections are typically administered in the lateral tail vein or retro-orbital sinus. IT injections... are “seeing” a great number of cells as they circulate in the blood, rather than being geographically...Abbreviations: AAV, adeno-associated virus; GRE, gene regulatory element; NHP, non-human primate. Many of...., 2023 AAV-PHP.V1* CNS: Biased toward vasculature, but also transduces astrocytes Mice Yes ...al., 2020 AAV-PHP.V2 CNS: Biased toward vasculature, but also transduces astrocytes Mice Yes ...(rats, infant green monkey, rhesus macaque); vasculature (adult marmosets), biased toward astrocytes (...

  20. 15 Hot Plasmids from 2017

    Type
    Blog Post
    ...Listen to the podcast segment on modulating iPSC reprogramming  Regulation of telomere length with TZAP .... Subscribe to the Addgene blog to get all the latest hot plasmid updates!     Tol2 Gateway-compatiable... 2015. PubMed PMID: 25772473 Targeting and manipulating interneurons using a viral approach Genetically...’s lab has developed a new AAV approach using regulatory elements that restrict expression to this cell...approach allows you to target and functionally manipulate GABA-ergic interneurons across species and will...combination of deliberate and random mutagenesis to isolate the best RFP. They targeted known residues on the...brightness, and proper maturation. Three new RFPs were isolated and characterized: mScarlet (fluorescent lifetime...

  21. RaPID Detection of RNA-protein Interactions

    Type
    Blog Post
    ...proteins that promote RNA decay and their upregulation is correlated with poor survival in breast cancer patients.... BirA* requires 16-18 hours to sufficiently biotinylate interacting proteins, which results in poor spatiotemporal...interactions with RaPID RaPID is similar to the BioID platform for identifying protein-protein interactions, ...promiscuous E. coli biotin ligase, BirA*, to biotinylate proteins that are within ~10 nm of the bait, ... also tweaked BirA* to increase its rate of biotinylation. The improved enzyme, called BASU, is derived...one to study RNAs expressed from their native regulatory elements. Controls for RaPID experiments RaPID...Affinity Purification (CRAPome), to identify highly biotinylated background proteins reported by other research...

  22. Transferable Skills Guide: Cross-team Communication

    Type
    Blog Post
    ...involved “plates,” and our scientists and lab technicians always knew from context what a “plate” was. Our...these plates now” to a fellow team member. But, at Addgene, we use seven different types of “plates” (Fig...barcode for your plate, you will get in trouble if you don’t qualify what kind of “plate” you mean. Without...to know what kind of plate they were talking about during the meetings. And “plate” certainly wasn’t the...our engineers (Fig. 2). Creating this guide was relatively easy. I took a picture of virtually every container...information Prescribing an official name to all of our plates, tubes, and racks meant we had fewer errors in ...important documentation, including SOPs or project-related documents, we define new and potentially ambiguous...

  23. CRISPR Meets Synthetic Biology: A Conversation with MIT’s Christopher Voigt

    Type
    Blog Post
    ...engineering projects in a very big way. In one of his latest papers, published in Molecular Systems Biology ...CRISPR/Cas genetic circuits are linked to the regulatory networks within E. coli host cells. ...in building genetic circuits is getting enough regulatory proteins that don’t interfere with each other...programmable that you can make a very large number of regulators. That basically means larger genetic circuits...that could conceivably be as large as natural regulatory networks. Addgene: What kinds of things could... pattern, shape. All of that is controlled by regulatory networks in the plant. If we want to access that...biology can do - it’s good to be able to build regulation thats as sophisticated as a natural network. ...

  24. Antibodies 101: The Basics of Western Blotting

    Type
    Blog Post
    ...protein’s post-translational modifications such as phosphorylation, acetylation, methylation and ubiquitination...proteins are being up- or downregulated in a system, detect post-translational modifications, quantify ...quantify protein levels relative to standards, detect the cellular location of proteins, and can be a readout...submerged in a tank filled with a transfer buffer formulated to conduct the electric current. The wet transfer...thickness of the band provides information about the relative abundance of the protein in the sample. More protein...immunosorbent assay (ELISA) (Aydin et al., 2015). Post-translational modifications In addition to protein abundance...ubiquitination. For example, to test if a protein is phosphorylated following a specific experimental condition...

  25. Plasmids 101: Restriction Cloning

    Type
    Blog Post
    ...colonies on the backbone alone plate and many colonies on the backbone + insert plate (or at least more colonies...backbone alone plate). Unsuccessful ligations will usually result in few colonies on both plates (unsuccessful...), in a vector alone plate with many more colonies than the vector + insert plate (unsuccessful 2), or... each plate (unsuccessful 3).     If you have a high number of colonies on your backbone plate (greater... Follow the manufacturer’s instructions.   2. Isolate Your Insert and Vector by Gel Purification     ...throw the digestion mixtures together. You need to isolate your insert and backbone from the enzymes used ...base pairs for each varies, it is difficult to calculate this based on DNA concentration alone. One method...

  26. Plasmids 101: Multicistronic Vectors

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    Blog Post
    ...Elements Translation in eukaryotes usually begins at the 5’ cap so that only a single translation event ...plasmid is packaged for viral delivery, or the relative expression levels between two or more genes is...single-stranded RNA viruses to allow for the efficient translation of multiple genes from a single transcript. The...Entry Site (IRES) to allow for initiation of translation from an internal region of the mRNA. In the ...discovered in poliovirus RNA, where it promotes translation of the viral genome in eukaryotic cells.1,2 Since...they all have in common is the ability to spark translation initiation independent of the 5’ cap.  IRES elements...bicistronic expression; however, many could be easily manipulated to express more than two genes. Plasmid Name...

  27. What's New in CRISPR - March 2020

    Type
    Blog Post
    ... via acetylation or demethylation. Casilio DNA Methylation Editing (Casilio-ME) is a methylation editing...human pluripotent stem cells CRISPR-based DNA demethylation Optogenetic repressors of gene expression ...50–90% of average on-target DNA-editing levels relative to BE4max.  Read the paper in Nature Biotechnology...Michael Laub’s lab developed a CRISPRi system to downregulate expression of specific Caulobacter genes using...ctrA, a gene encoding an essential cell cycle regulator. The lab expressed both dCas9 and the ctrA sgRNA... demonstrated the system can also be used to downregulate multiple genes at once. Read the paper in mBio...Find the plasmids Enhanced CRISPR-based DNA demethylation Figure 2: The components of Casilio-ME1...

  28. Plasmids 101: Choosing an Antibiotic Resistance Gene

    Type
    Blog Post
    ... resistance genes so that you can specifically isolate bacteria containing your constructs of interest...bacteria as a way to produce more of the plasmid, isolate individual plasmids, or express a gene contained...goal of your experiment — to grow, express, or isolate plasmids. The presence of an antibiotic resistance...shaking at 37 degrees) can be shortened since the plated cells will have time to produce beta-lactamase ...to produce satellite colonies when used on agar plates due to the fact that beta-lactamase is excreted...Cons: Prone to satellite colony formation on agar plates, less stable at high temperatures and in acidic... and is less susceptible to degradation in agar plates. This prevents satellite colonies from forming ...

  29. Plasmids 101: Colony PCR

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    Blog Post
    ...highlighted below. Preparing template: Pick a single colony with a sterile flat toothpick or pipette tip ... clones for later culture: At this point, you will want to hang onto your clones for later use. There ..., just streak the colonies on an LB plate. You can use this plate to start liquid cultures. Lastly, you...polymerase) using the supernatant of lysed bacteria as template; and 3) run your PCR product on a gel to analyze... to preparing a standard PCR reaction: combine template, primers, polymerase, and dNTPs and then incubate...released from the bacteria in order to serve as PCR template. Dealing with this and a few other colony PCR ...background colonies on your no ligation control plate. The more background, the more colonies you will...

  30. Deep Dive: Statistical Tests (Comparisons)

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    Blog Post
    ... vs. the factor that is being experimentally manipulated. Below are several core factors of statistics...t-tests Samples that come from the same subject or population - before and after a treatment, for example -.... Samples arising from different subjects or populations (a treatment group vs. a control group), on the...analysis, must be performed. These will be discussed later. One and two-way ANOVAs ANOVA tests can compare...Quantifying and controlling for any covariance between manipulated independent variables allows for a more accurate...how independent variables you are not directly manipulating affect an outcome (age, demographic, diet, etc...to know every pairwise comparison within your population? Sometimes this can’t be avoided, but if it can...

  31. Hot Plasmids: Fall 2024

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    Blog Post
    ...furimazine. Right: Demonstration of PILATeS. Cell adhesion to a PILATeS-functionalized coverslip creates ...reversible, making PILATeS suitable for live imaging with a time resolution of minutes.  Find PILATeS plasmids ... F., Goswami, K. K., & Russell, W. C. (1987). Isolation and characterization of monoclonal antibodies ...antigenic differences between human, canine and simian isolates. J Gen Virol., 68 (Pt 11), 2769–2780. https://... happen within a few hours. In order to use the latest mScarlet tags, mScarlet3 and mScarlet-I3, Oliver...for CRISPR/Cas9 in M. abscessus here! Neo D, Clatworthy A, and Hung, D. (2024). A dual-plasmid CRISPR...Bioluminescent reporting of mechanical forces with PILATeS By Emily P. Bentley Time to get flexible! Alex ...

  32. How to Be an Excellent Trainee

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    Blog Post
    ... learned, and ask questions later – maybe even the next day. In the latter case, you can say “I usually...planning you can do to build a bench mentor-mentee relationship and bring your best self to the training experience...plenty of articles on building successful mentor relationships, asking for mentor support, and choosing your...the research group). The bench mentor-mentee relationship is extremely important – without a good set ...mentor than your scientific mentor. Thus, this relationship will probably define your experience in the ...Luckily, you can do a lot to contribute to the relationship dynamic and set yourself up for success! Getting...ll probably forget and end up asking your mentor later if you don’t write it down. And don’t be afraid ...

  33. Antibodies 101: Introduction to Antibodies

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    Blog Post
    ...chemiluminescence, precipitate the protein out of solution, or isolate cells expressing this protein. Read on to learn...antibodies against the injected protein. Several weeks later, blood from the animal is drawn and the antibodies... hand, monoclonal antibodies are a homogenous population of antibodies that all recognize the same epitope...dilution, which produces an immortalized, clonal population of cells that produce a single variant of antibody...antibodies, recombinant antibodies are a homogeneous population. However, unlike monoclonal antibodies, which.... Western blots are frequently used to compare relative levels of protein expression between cell types... present. The reaction takes place in a 96-well plate, where primary and secondary antibodies are used...

  34. Five Popular Model Organisms, Part 2

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    Blog Post
    ...biologist tool box” for its common use in plasmid manipulation and genetics research. Plasmids are ideal tools...studying genetic modification for their ease of manipulation and their ability to be replicated. Because ...processes naturally found in E. coli have been manipulated for use in molecular biology studies. For example...BSL-1 status, E. coli can be used as a model to relate these processes in more pathogenic bacteria (Blount...rat, is another popular model organism. A close relative to the mouse, both are great mammalian models ...limiting attributes of rats is their difficult to manipulate genome and limited genetic data. To bridge this...biological processes and signaling pathways that regulate regeneration and wound healing are conserved across...

  35. Antibodies 101: Polyclonal Antibodies

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    Blog Post
    ...immunogen, then isolating and purifying the antibodies produced from its serum several weeks later. Created ...as KLH or Freund’s adjuvant is injected, which stimulates the overall immune response. Once the initial... antibody response starts to wane about a month later, the animal is given additional booster immunizations...contains the antibodies - is further processed to isolate the antibodies. Most commonly, antibodies of the...the IgG isotype are used for research purposes. Isolating polyclonal antibodies from the serum There are...are a few different ways the antibodies can be isolated from the serum. Protein A/G purification Protein...different type of purification can be performed to isolate antibodies only recognizing your protein of interest...

  36. The time and cost required to make a plasmid

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    Blog Post
    ... overall inflation of USD has gone up 19.8% in the same time period. For ease of calculations*, let’s ...well (or 384-well) plate! How much do repositories help?   Designing precise calculations for any of the ...Debate of ’18, Blue Flame depositor John Doench calculated the time to make a plasmid. Doench’s lab in the...long does it take in total? While much harder to calculate, in both our 2018 and 2023 poll, most people said...much does it cost to make a plasmid? Doench also calculated the cost of consumable reagents used to make ...2018 numbers, and the intervening five years, inflation has occurred. Here at Addgene, the consumables...s split the difference and put scientific inflation at… roughly 34.9%, which means that the same plasmid...

  37. Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP

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    Blog Post
    ...reporter lines for more sophisticated experimental manipulations, saving the time and money needed to develop...control gene expression, he could then selectively manipulate only GFP-labeled cells. Once Tang and Cepko found...but specific system. T-DDOGs also successfully regulated expression of channelrhodopsin-2, commonly used...for optogenetics experiments in specific cell populations. Listen to Our Podcast Interview with Connie ...-DOGs The T-DDOG results showed that GFP could regulate transcription in a cell-type specific manner, ...Cepko were eager to see if they could apply GFP regulation to other types of proteins. They started with...system may make optogenetics in specific cell populations even easier. Advantages and other possibilities...

  38. Addgene's Cancer Collection Pages Connect the Research Community

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    Blog Post
    ...specific genes involved. Downregulation of tumor suppressors and upregulation of proto-oncogenes ­­­can...while simultaneously stimulating growth and division. The combined effect is unregulated proliferation. Another...cases arise and millions of people die from cancer-related complications each year. Despite these grim statistics...cells still need to successfully infiltrate the circulatory or lymph systems to spread, or metastasize. Many...Many cancerous cells will die trying, as the circulatory and lymph systems are not ideal environments ...Ras researchers, one of the most exciting and relatively new resources is the RAS Pathway Clone Collection...set of vectors for use with the Gateway Cloning Platform (Life Technologies, Carlsbad, CA) that represents...

  39. CRISPR 101: Making a Knock-In Cell Line

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    Blog Post
    ...knock-out. The goal of a knock-out is to disrupt DNA translation by generating a frameshift mutation. The majority...like to introduce the edit. Finally, you must stipulate what sequence to introduce, which is done through... will guide the cell to use the donor DNA as a template for repair, which will result in the incorporation... knock-in! So how does the donor get used as a template for repair? Read on to find out! Homology-directed...is recommended you check your cell lines for HR-related mutations before attempting knock-ins by HDR. ...basis of the HDR pathway relies on repair from a template molecule, which is usually an endogenously available...and uses the ends of the ssDNA to find a repair template. The highest HDR efficiency is observed when inserts...

  40. Simplify Cloning with in vivo Assembly

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    Blog Post
    ...enzyme specifically cuts methylated DNA, so it will selectively destroy the template, but not newly synthesized...-55 °C for the homologous region (calculated using Oligo Calculator). Insertions - To insert short sequences...confirm primer design.  Use minimal template DNA Use 1 ng of PCR template DNA to minimize false positive colonies...for example for subcloning genes from different templates. All PCRs are run as an 18 cycle, 25 μl single-tube...Primers consist of two regions: the 3’ end binds template DNA for PCR amplification, while modifications...sequences are encoded at the 5’ end. First design the template binding region, with a melting temperature (Tm... bp, such as epitope tags, design primers with template binding regions to amplify away from the insertion...
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