We narrowed to 801 results for: DES

...ultracompetent (109 CFU/μg) cells. Primer design Using specific primer design (Figure 2), IVA cloning can be used...primers that bind at either side of the undesired portion, designed to amplify the rest of the vector sequence...primary method to produce the linear fragments, as desired modifications and homologous regions can be encoded...specifically cuts methylated DNA, so it will selectively destroy the template, but not newly synthesized sequences...mutagenesis to complex assembly of multiple genes. Primer design for each type of modification is shown in Figure... main requirement for IVA cloning is carefully designed primers. The majority of mistakes are due to incorrect...homologous sequences are encoded at the 5’ end. First design the template binding region, with a melting temperature...

...step to colony PCR is designing primers. There are 3 strategies for primer design: 1) insert-specific primers... second option is to design backbone-specific primers. These primers are designed to anneal to sites that...less money. The key steps to colony PCR are: 1) design primers to detect the presence of your insert; ...things to consider when performing colony PCR.   Designing colony PCR primers The first and perhaps most ...-specific primers: Insert-specific primers are designed to anneal to an insert-specific sequence. This...backbone but that contain different inserts. When you design primers to anneal outside the cloning site, it ...about insert orientation, then you might consider designing orientation-specific primers. Blunt end cloning...

...working with CRISPR than just designing a repair template that contains your desired edit, and I want to make...say about CRISPOR, a tool to help you design, pick, and close guides for Cas9 and Cpf1. This tool supports...The blog post I mentioned, John Doench’s How to Design your gRNA for CRISPR Genome Editing, gets over ...these numbers show that this basic concept of gRNA design is still challenging for researchers using CRISPR..., and that standardizing and improving the gRNA design process and rules for various applications is necessary...tackle the challenges of DNA repair (with bonus gRNA design resources.) Subscribe to CRISPR updates on our ...in Human Cells Hands-on tips for repair template design from guest bloggers Dominik Paquet and Dylan Kwart...

...DNA guides NgAgo uses 5’ phosphorylated DNA guides (so called “gDNAs”) rather than the RNA guides employed...24 base DNA guides may be more user-friendly, since they can be ordered as oligonucleotides and should ...gDNA guides will also need to be transfected into the cells of interest. In contrast, RNA guides must ...important role in RNA interference, binding RNA guides to cleave foreign RNA. Argonaute proteins are also...cleave DNA in vitro, directed by phosphorylated DNA guides. Unfortunately, Swarts et al. were unable to show...NgAgo preferred certain guide sequences - using 5 guides/gene for 10 genes, they did not observe differences...a different approach, randomly removing 1-20 nucleotides from the cleavage site specified by the gDNA....

...for CRISPRi are described here: http://www.addgene.org/crispr/qi/ Plasmids described in  Zalatan et ...stepwise protocol on designing and using RNA scaffolds: Delebecque, C. J. et al. Designing and using RNA scaffolds...regulators allows a greater degree of control of desired expression levels dependent upon the type of transcriptional...transcriptional regulator or the number of designed scaffold binding sites. With hundreds of characterized...tunability of transcription is possible. From a design perspective, this is appealing since the RNA scaffold...pairing, allowing for rational, straightforward design. Since the technique relies on base pairing, it...and multidirectional multiplex control via scRNA design is readily apparent in metabolic engineering. The...

...shipping. However, there were issues that persisted despite our best efforts. With a little over half of our...packages navigated customs and arrived at their destination in a timely manner and at the correct temperature...for their experiment.     Table 1: Most popular guides, protocols, and collections of 2023. Educational...Educational Resource Top Five by Views Guides CRISPR Guide Lentiviral Guide AAV Guide Optogenetics...Electrophoresis Bacterial Transformation How to Design a Primer Creating Bacterial Glycerol Stocks Inoculating...Origin of Replication What is a Plasmid? How to Design Your gRNA for CRISPR Editing The Promoter Region...Analysis Videos What is a Plasmid? How to Design Primers for PCR Agarose Gel Electrophoresis Inoculating...

...your genome edits, this paper provides detailed instructions on how to design such assays, in addition to...great deal of precision. This means digital PCR provides an absolute measure of target abundance, does ...what kind of assays can be used to sniff out your desired mutations. The assays you will use aren’t that ...often used in real time PCR. These assays can be designed to detect either a donor sequence integrated through...non-homologous end joining (NHEJ) (2-5), depending on your desired genome edit. Since exploitation of NHEJ to generate...the target locus, as well as a reference probe designed to bind at an unedited site distal tothe predicted...double strand break, and a NHEJ/ “drop-off” probe designed to bind directly at the predicted double strand...

...essentially any DNA sequence desired. This technology allows you to create a pair of designer proteins that will...nucleases that will bind desired sequences. TALENs are much more reliable. If you design them for a particular... simple to design TALENs. It only takes two to three days from the moment you are designing a TALEN on...Cell, Musunuru, Chad Cowan and their colleagues describe a much more efficient tool for doing that disease...people unfamiliar with this new TALEN kit, can you describe it for us? Musunuru: TALENs are a type of genome...target a particular DNA sequence? Musunuru: The kit includes 832 plasmids containing prefabricated pieces that...to build a DNA-binding domain to recognize any desired 15 base pair sequence in the genome in rapid fashion...

...screening by PCR may be the way to go. Design a PCR to flank your desired edit site and run the PCR product...nucleotide along with your desired edit. This silent mutation should be designed solely for restriction enzyme... updated Nov 3, 2022 by Susanna Stroik. You’ve designed your gRNA and introduced it into your target cells... your cells were appropriately edited for your desired mutation - insertion, deletion, or site-directed...know a few things about your target cells and the desired edits. First, you will need to know what the ploidy...knockout mutations When a knockout cell line is desired, any mutation that introduces a frameshift in the...frequency of your sgRNA-Cas9 combo. Pro tip: When designing your PCR, ensure there is at least ~200 base pairs...

... later this year. Designing the donor plasmid Figure 2: Donor plasmid design strategy. A donor ...with each cell line. The editing strategy and design described above can be used to create similar donor ...GFP tag and 1kb of homologous DNA flanking both sides (about 2.7kb total)—is inserted into a plasmid backbone...plasmid to prevent the plasmid from being cut and destroyed by CRISPR/Cas9 during transfection. Making the...use electroporation, a technique that briefly destabilizes the cell’s outer membrane and allows the components...Addgene’s CRISPR-based Protein Tagging collection includes systems for tagging mammalian cell lines, Drosophila...

...and should be designed accordingly. If you are trying activate a gene, you should design a control that...carried out by scientists every day. Although the designs and outcomes may vary, one thing should be present...these control plasmids are critical for the correct design of the experiment.  The Experiment: Knockdown the...control plasmids Part of planning your experiment includes determining what factors need to be controlled...microscopy images resulting from our experiment that includes the aforementioned internal control and is consistent...correct interpretation of the results because it provides an important reference point when analyzing the...positive control, the internal control vector, was described earlier. Once you are sure your conditions are...

...overall HDR rates, we have shown recently that desired genome-editing events by HDR can be generated more...genome editing with CRISPR/Cas9 in human cells was described in 2013 (Cong et al, Mai et al) we were very excited...introduced. Although we were able to introduce the desired mutations in genes associated with dementia, such...CRISPR from re-cutting the target sequence once the desired edit has been introduced. We found that blocking...only saw incorporation of the blocking mutation. Despite the fact that both the blocking mutation and our...thinking further about the distance relationship described above, we realized that, in addition to increasing...efficiency a guide RNA targeting a cut <10 bp from the desired mutation should be used. For a heterozygous event...

...different ways. We describe a number of issues with some of the experimental designs used to make these...of Amsterdam. The previous post in this series described a practical approach to selecting a bright fluorescent... protein. Photobleaching is the irreversible destruction of a fluorophore under the influence of light...photobleach at some point. For live-cell imaging, it is desirable to have fluorescent proteins that are photostable...photoswitching (Kremers et al, 2009), which usually are undesired properties. In the ideal situation, a fluorescent...large number of emitted photons before they are destroyed. Factors that affect photostability The photobleaching...fluorescence, which, in its simplest form, can be described by a mono-exponential decay (analogous to radioactive...

...SapTrap, the user first designs either oligos or synthetic DNA for the desired gRNA target sequence, as...specified by a guide RNA (gRNA) sequence. Users can design gRNAs to induce the break at a specific genomic...need for PCR or cloning, as digestion of the destination vector with SapI yields 2 sites- the first site...accepts the homology arm repair template. SapTrap includes a prebuilt donor plasmid library containing several... and a Cas9 expression plasmid will insert the desired genetic tag and marker sequence into the targeted... a tissue specific manner, as well as 3-site destination vectors for inserting a tag at multiple target... not work with C. elegans, several groups have designed modular toolkits to assist with tagging genomic...

...the order of nucleotides in a string of DNA (7). Sanger sequencing uses modified nucleotides, called dideoxynucleotides...the widespread use of Sanger sequencing. Building upon this technology, radiolabelled nucleotides were...causes sickle cell anemia. The mutation happens to destroy a sequence recognized by the restriction enzyme...synthesis reaction along with all four regular nucleotides results in the synthesis of DNA strands of all...fluorescence activity as fluorescently labeled nucleotides are incorporated, a method called pyrosequencing...nanopore-based DNA sequencing which reads the nucleotides of a DNA strand as it slips through a tiny pore... the different electrochemical properties of nucleotides (8). Compared to older technologies nanopore-...

...Have you ever designed a CRISPR guide RNA and wondered why it is limited to only 20 bases, or why it’...understand the parameters of CRISPR experimental design. Let’s dive into some structural biology! Major... lobes, REC and NUC, and their subdomains. NUC includes the HNH and RuvC catalytic domains, as well as...as the CTD (also known as the PI), while REC includes most of the bridge helix. Created with BioRender.com...Fact! Even though the Cas9:RNA complex is well described in the closed conformation, recent electron microscopy... does not include stem loop 3, and guide RNA nucleotides 11-20 are not resolved due to structural disorder...2017). Within an interior cavity, Cas9 grips nucleotides 1-10 of the guide RNA, prearranged for base pairing...

...from Wong, 2018   Primers Design of primers and probes Primer design, of course, could easily be its...useful primer design resources in the References and Resource section. Validation Once designed, your primer.... For example, chemistries that rely on hairpin design of probes/primers often offer higher allelic specificity...Taqman) Low; can be reduced with a double-quencher design Yes, one Yes Yes Agarose gel or uMelt analysis...Reference Genes  Choosing a set of reference genes deserves more attention than it usually receives. Different... multiple reference genes whenever possible (Vandesompele, 2002), and it's possible to multiplex your ...documented in the literature, a geNorm study (Vandesompele, 2002) will help you select the appropriate ...

...this article, Quilt CTO Aneesh Karve, shows how to design experiments that work anywhere in the genome. Aneesh's...enhancers! This leads us to a three-step approach for designing an experiment to identify the critical NANOG binding...Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen to target and disrupt the NANOG...anyone can find enhancers with Quilt. Seek and destroy enhancers with CRISPR Armed with the genome math...expression for NANOG enhancers, we’re ready for step 2: design a CRISPR screen to disrupt these enhancers. The...attenuated retrovirus in the same family as HIV. By design, our lentivirii are genetically programmed to CRISPR...Lentivirus from Addgene If you’re interested in designing your own CRISPR screens for enhancers, check out...

...many MoClo kits are designed with specific model organisms in mind, others are designed for flexibility. ...Synthetic biology in bacteria MoClo is commonly used to design TUs and multi-TU constructs for synthetic biology..., biofuels, and natural products without the undesirable side products generated during chemical synthesis.... phaffii, then the MoClo YTK Kit specifically designed for S. cerevisiae is at your service (Lee et al...chromosomal integration and gene expression, the system includes a toolkit to express recombinant proteins within... are endless: genetic circuits with multiplexed guides, mini screening libraries, direct edit optimization.... The Fragmid kit described above works in both mammalian systems and Drosophila. Similarly, the Multi-Kingdom...

...their specificity or from the thymus to the lymph nodes. These methods gave researchers much more information...expressing cells. For example, by harvesting lymph nodes at different times after antigen challenge, scientists... cellular dynamics in lymphoid organs. In lymph nodes, for example, two-photon microscopy allowed scientists...extravasate from the bloodstream to invade lymph nodes and scan antigen presenting cells, but two-photon...interactions in diverse tissues, including lymph nodes, brain, liver, gut and skin. Two-photon intravital... 2016)? How do dendritic cells migrate to lymph nodes to activate T cells (Celli et al 2008, Kitano et...dynamics within the germinal center of murine lymph nodes. However, one must pay attention to time scale when...

...affects all kingdoms of life. Of course, that includes you. Besides the risk to the researcher, Zeocin has been...most yeast vectors and many mammalian vectors are designed to shuttle between species. Figure 1: Shuttle...host’s replication machinery. A shuttle vector designed for unrelated bacteria should include oris for...target species. Fun fact! Plant vectors are usually designed to shuttle between E. coli and Agrobacterium tumefaciens...protects against any of them. But different aminoglycosides work on different host species: kanamycin targets...media that supplies the key nutrient. The vector provides the missing synthesis machinery, and the cells...image for individual analysis. A shuttle vector designed for this approach in a mammalian cell experiment...

...Patrick Miller-Rhodes from the University of Rochester Medical Center. Patrick Miller-Rhodes is currently...This post was contributed by Patrick Miller-Rhodes, a Ruth L. Kirschstein NRSA Predoctoral Fellow at ...this technological gap, a trio of recent papers describe the development of CRISPR activation (CRISPRa)... large size of Cas9 activators, which largely precludes delivery by adeno-associated vectors (AAV). This...packaging capacity than AAV. Such an approach was described recently by the Day Lab, who developed neuron-... selected the immediate-early gene Bdnf, which encodes brain-derived neurotrophic factor (BDNF). Bdnf ...High-throughput screening strategies Although the tools described above make CRISPRa more accessible to neuroscientists...

...reproducibility than others. Unfortunately, most ELNs are designed as closed ecosystems and act as data silos because...RSpace ELN supports reproducibility RSpace was designed in response to requirements that emerged from ...used in research, and data repositories. Below I describe how each of these kinds of connectivity facilitates... in institutional and lab servers specifically designed to hold large amounts of data. Although it may... use of other tools they use in research. This includes chat apps like Slack, Microsoft Teams, and Google...individual documents or more complex datasets into the desired repository. They also support entry of required...deposits will be made. The Dataverse integration is described in this video and the Figshare integration in ...

...property by including ccdB on its destination plasmids. In the destination plasmid, the ccdB gene is flanked... rest do not grow. While there are many ways to design your positive selection strategy, here are a few...straightforward in cases where the inserted DNA includes an antibiotic resistance marker. After transformation...grow. However, often times, the plasmid backbone includes an antibiotic resistance cassette and you aren...backbone contains your gene of interest. Auxotrophy Besides antibiotic selection, you can select for a successful...prototrophy come into play during cloning if you’ve designed your experiment using an auxotrophic strain of...sites. To introduce a gene of interest into the destination plasmid, recombination sites must first be added...

...Aptamer Fluorophores describes the enrichment process used to evaluate oligonucleotides for binding affinity...of Spinach. In doing so, they identified the nucleotides that contribute to the instability of Spinach...similar levels of fluorescence when bound to DFHBI despite being approximately half the length (Warner et ...also smaller than any form of Spinach - only 49 nucleotides in length - and is better able to maintain its... of colors, so do aptamers! Corn was recently described in 2017 (Song et al., 2017). Corn naturally dimerizes... Red and orange fluorescent aptamers Corn was described in the same study as Red Broccoli and Orange Broccoli...Broccoli, and share similar properties to Broccoli despite binding to a different small molecule. Notably,...

...for the desired modification. Finally, the positive clones are sequenced to confirm the desired modification...Primer Design As a rule of thumb, 11 bp of complementary sequence on either side of the desired mutation...introduced to plasmids using primers (with the desired mutation) in a PCR protocol that amplifies the ...mutated clones. Forward and reverse primers are designed to be complementary, but each primer may extend...Polymerase To ensure that you don’t introduce undesired mutations through the PCR process, you need a ...The resistance marker from the parental plasmid provides a mean for selecting for transformants which have...mutagenized plasmids Once constructs containing the desired modification have been identified (and confirmed...

... Addgene plasmids by gene here), designing primers with your desired mutation, performing PCR to generate...multiple fragments of DNA to create a synthetically designed DNA sequence. There are multiple methods of DNA...purification steps and end-repair steps with well-designed fragment overlap sections, while still creating... guide you through both experimental and primer design based on fragment sequences and the polymerase ...enzymes are adjusted. Appropriate fragment and primer design results in covalently joined, scarless DNA – perfect...by assembling multiple fragments containing the desired mutations. This same process can be used simultaneously...oligo that contains a region of homology and any desired edits to the target region. Assembled plasmids ...

...express a gRNA designed to direct Cas9 to their target sequence of choice (see tips for designing a gRNA) and... of the major CRISPR lingo and will be able to describe the functions of the various CRISPR/Cas9 components...responsible for processing foreign DNA as well as the RNA guides required for endonuclease function, when used for...Bind to a guide RNA The guide RNA, which will be described in more detail below, enables Cas9 to cut a specific...DNA damage is a DSB within the target DNA ~3-4 nucleotides upstream of the PAM sequence. Cas9 Species/Variants...with the appropriate PAM sequence) to modify the desired genomic locus.   Some historical notes CRISPR ... combined with its adjacent spacer, ultimately encodes a single crRNA. The direct repeat regions contain...

... is also documented. Experimental design When you are in the design phase of your experimental planning...CasPEDIA is an online, searchable resource that provides summary information on Class 2 Cas enzymes. The...The information is organized in wiki format and includes biochemical properties, previously established...relevant resources The homepage displays a graphic describing CasIDs and relevant properties of Cas enzymes...What other RNA components are required? CasPEDIA provides information on crRNA and tracrRNA requirements... also show you how multiplexed arrays would be designed, if that’s your end goal. CasPEDIA also features...search (trans activity, multiplex ability, gRNA design, etc.). With this feature, you can compare different...

...diversification of all nucleotides at a user-defined loci in bacteria. Its modular nature provides versatility in...provide a means to continuously diversify all nucleotides within a user-defined genomic region. EvolvR,...method developed by the Dueber Lab at Berkeley, provides a new approach for generating novel genetic variants...users to achieve targeted diversification of all nucleotides within a defined region of a genome. Require...turbidostat. Only mutate particular types of nucleotides. Both the Bassik Lab and the Chang Lab have created...3’ end, while its native endonuclease activity degrades the displaced strand.   Figure 1: EvolvR...single starting cell. Additionally, multiplexing of guides allows EvolvR to target more than one genomic site...

... error rate for long sequence knock-ins Visual barcodes for live cell clonal multiplexing Improving ...nanobody-GFP interaction). The modularity of the system provides researchers better flexibility and makes the approach...other experiments. And, while the BLITZ system was designed and optimized with zebrafish in mind, it can easily...https://doi.org/10.1038/s41556-021-00836-1 Visual barcodes for live cell clonal multiplexing by: Rachel Leeson...Recently, Ravid Straussman’s lab has developed visual barcodes for clonal multiplexing of cells in live imaging...peptide. By combining three different localization peptides with four different fluorescent proteins, and ...freeware.    Fig. 4: Images of 12 visual barcodes in use in A375 cell line. Image from Kaufman, ...

...site by the pegRNA — a guide RNA that also encodes the desired edit and homology to the genomic DNA locus...both specifies the target site and contains the desired edit (or edits) engages the prime editor protein...and the reverse transcriptase domain copies the desired edit, directly polymerizing new DNA onto the nicked...edit within what Fyodor Urnov refers to as “PAM deserts” in the genome. More versatile and precise than...editors are preferred. For instance, if target nucleotides are positioned within the canonical base editing...still almost always results in higher ratios of desired edits to indel byproducts than Cas9-initiated HDR...different constraints and affect how you select and design your prime editing tools. Here, we’ll briefly cover...

...cloning, or MoClo, is a systematic cloning strategy designed by synthetic biologists to perform large scale...specifically refers to the organizational schema described above, and not any particular cloning technique...example of an individual part, this category also includes promoters, UTRs, terminators, etc. Once selected...behave in a cell. For unpredictable cases, MoClo provides two answers: first, sets of parts with characterized...protein products can interact with the DNA that encodes the protein itself or other genes within the circuit...cellular engineering goal, several of which are described below. Multigene circuits assembled through MoClo...for more complex CRISPR experiments (e.g., many guides/parts delivered) in certain model organisms. In...

...A recent publication [1] from the Sánchez lab describes the plasmids and protocols required for both WISH...companion resource called “cutting class” which provides more detailed materials and protocol resources...synthetically made RNA (riboprobes) with modified nucleotides (for example, Digoxigenin-labelled Uracil) that... molecular cloning to insert the cDNA into the desired plasmid backbone. The Sánchez lab plasmids allow... which are available through the deposit) are described in the Sánchez lab publication [1] so that the...expressed mRNAs.These native mRNAs are subsequently destroyed by Argonaute-catalyzed nuclease activity or their...have to use a commercial E. coli strain that is designated as DE3. For Phagocata morgani and Phagocata gracillis...

...for and how can you do it well? Below, I briefly describe some tips for a successful postdoc fellowship....typically writes the grants that fund your research, guides the research directions of the lab, gives you constructive...Thesis committee members provide expertise in designing and interpreting experiments, as well as career... there a tractable question or are you simply describing a phenomenon? Exploration can be important, but...presentations benefit from practice. If you simply recycle slides from the last time you gave a presentation to a...story I can tell with my data?  Can I make better slides that more effectively tell a story? Generally,...or functional models or more helpful background slides. If you don't have much new data, speculate on ...

...extremely useful if a desired protein product is toxic to cells at high concentrations. Despite their advantages...and induced by lactose, provides the highest level of protein expression. Despite their ease of use, it...for easier access and purification of a desired protein. Despite being a rather nascent technology, plant...for proper protein function. Most DNA constructs designed for mammalian expression utilize viral promoters...protein in bacterial cells: the pET, pRSET, Gateway pDEST, and pBAD vectors for example. Protein expression... include ribosomes, RNA polymerase, tRNAs, ribonucleotides and amino acids. Cell-free expression systems...

...Plasmid purpose: This is a brief description of what the plasmid was designed and used for in the depositing...plasmid, how will it be sent to me? Description: This describes how the plasmid or other product (kit... Gene/insert: This describes what has been inserted into the backbone, and provides information about ...successful cloning. Cloning information: This section provides useful information about how the vector was assembled... sequencing. Growth in Bacteria: This section describes how to grow the bacterial strain in which the ...relatively new feature of our plasmid pages, and provides a list of publications which have used the plasmid...

...reporters are challenging to design and difficult to multiplex. The Covert Lab provides an alternative tool with...fluorophores can be multiplexed together. Challenging  to Design: FRET reporter activity depends on both the distance...advantages. First, they are easy to multiplex. Their design allows flexible swapping of fluorescent proteins...overlap are used together. Second, KTRs are easy to design. The key determinants of KTR function are the positions...See Box 1 in Kudo et al. for more details on the design of the bNLS and NES sequences. Disadvantages of...translocation rates are another variable to consider when designing KTR experiments. Third, KTRs are not well suited...JNK, and ERK, or a backbone to use for custom KTR design, plasmids are available from Addgene! References...

... Zimmer provides several. In Part Three of the book, called simply “Old Science,” he describes the now...teaching and communicating science for over two decades, attempts to answer it in five paragraphs, with...recent resurgence of “Do-It-Yourself Science” described in Chapter 3, and of course the rapidly changing... science.”  The three chapters in this section describe the challenges of trusting science in a “fake ...changed significantly over the last decade. He describes the detection of gravitational waves--a story ...collaboration from scientists around the world. The paper describing the waves’ detection had over 1,000 authors. ...This last section of the book, Future Science, describes the author’s thoughts on science’s biggest challenges...

...These libraries utilize optimized sgRNAs that were designed based on the rules identified in Doench et al....1,000 non-coding control sgRNAs. This library was designed to improve on-target activity while reducing off-target...with 6 sgRNAS, highlighting the benefit of sgRNA design in CRISPR libraries. Thus, the Brunello library... that if an essential gene plays a role in the desired screening phenotype, it may be missed because it's... the sgRNA metrics, confirming again that sgRNA design plays a significant role in library efficiency....CRISPRa, sgRNAs were targeted between 150-75 nucleotides upstream of the TSS. Similar to the Brunello ...Activation Mediator) uses an improved activator design that consists of inactive Cas9-VP64, a sgRNA with...

... about! You could describe your organizational system, your rationale for designing it the way you did...experience and skills would help you do the job as described. Let’s imagine you’re applying to a job that ... your cover letter, you can expand on that by describing how you developed and used a ddPCR assay to answer...question. Now, the hiring manager knows that you can design experiments with ddPCR, interpret the results, ...start by looking at the requirements in the job description. For each requirement, ask yourself if you have...to their work. Since the beginning is often the hardest part, here are some examples to get you started...company/organization]. I am a [adjective that describes you] scientist with over [number] years of experience...

...vectors and design your sgRNA targeting sequence using one of the many freely available sgRNA design programs...many cases several exons’ worth of DNA. Any ∼20 nucleotides of DNA can serve as the target for Cas9’s single...Cas9 localizes the deaminase to where the edit is desired, and it performs the programmed edit within the...flexibility. A consideration with this tool is that the desired edit will be performed on all relevant bases within...system utilizes an RNA template containing the desired edit adjoined to the sgRNA, a reverse transcriptase...not require an expertly positioned PAM without undesired bases nearby that may undergo unwanted editing....   Ok, I have a Cas9! What do i do next? Designing or selecting a sgRNA Addgene may carry a validated...

...Researchers have used oligonucleotides in fluorescent visualization techniques for decades, most notably for... exponential enrichment (SELEX), some 1010 oligonucleotides could be screened for their affinity to a ...aptamers as well, and over the course of the next two decades, these nucleotide-based ligand binders would prove...their broad range of targets, including proteins, peptides, amino acids, drugs, metal ions, and cells (even...counterparts, antibodies and enzymes, such as: Design and selection for targets can be done largely in...such techniques can be problematic, however. Well designed aptamers can help reduce such background, thereby...relatively bulky. One of the advantages of oligonucleotides over proteins is their modularity, which is...

...contamination and match their original description. The oldest, largest, and most diverse of these repositories...culture in the present day Cell culture is a widespread tool used in the fields of oncology, virology...problems surrounding cell culture over the last few decades some pharmacologists have tended to avoid cell ...International Cell Line Authentication Committee includes a database listing all cross contaminated or misidentified...comparable studies to be performed on a cell line even decades after it is initially isolated. Regrettably this...Master and Working stocks to work from. The future Attitudes are changing but yet more is needed to convince...can be prevented and we can progress faster to desired clinical endpoints.   Nick Amiss is an ATCC Outreach...

...Good as a WFH desk, and as a video control center.   Cycle 2: Zooming Forward Design: We produced ... So, much like SynBio, it was time to start our Design-Build-Test-Learn cycles. If you’re not familiar...again. Here’s that process!   Figure 1: The Design-Build-Test-Learn cycle adapted for video making...through. I know cameras, but we needed someone else. Design: Jennifer thankfully volunteered, and we decided...here, the operator is in a different room.   Design: The previous cycle made me realize we needed to...restrictions in place, most importantly, masks.  Besides the muffled voices beneath masks, humans rely on...reach for Addgene.  So, onto research! I needed to design a system that would allow me to set up a camera...

...Addgene’s catalog is a new class of sensors for neuropeptides and neurotransmitters. Motivated to develop ...GPCRs recognizing various neurotransmitters, neuropeptides and other ligands - almost A to Z! - from ATP...a new preprint, Jared Toettcher and colleagues describe pYtags, a modular system for monitoring specific... tool for monitoring RTK signaling.  Figure 4: Design of pYtag fluorescent biosensors for monitoring ...iGECInano plasmids here!   Matlashov ME, et al. Design and Initial Characterization of a Small Near-Infrared...intermediate affinities from 40 to 140 mM. These new designs greatly broaden the options for potassium biosensors...further engineering. Figure 5: Structure-guided design and genome mining both led to new sensors with ...

...suggests there are hundreds. This only includes general lab strains designed for subcloning or protein expression...was updated on Nov 14, 2017. You've worked hard designing your plasmid – you carefully selected the optimal...Dr. Theodor Escherich, the scientist who first described them in 1885. E. coli are mainly found in the ...commercial lab strains of E. coli used today are descended from two individual isolates, the K-12 strain ...mutations found in E.coli strains Gene(s) Description Functional Consequence dam DNA adenine methylase... fertility plasmid. A low copy-number plasmid, encodes proteins needed for bacterial conjugation. Genes...resistance to tetracycline Additionally, Table 2 provides a quick reference for some of the popular strains...

... Just as physical barcodes help librarians track individual books, genetic barcodes allow researchers ...to identify specific cells — as well as their descendants. This makes them useful for lineage tracing, ...pMuSIC library, or by sequencing the unique DNA barcodes. Figure 1: Schematic of barcoding with ...CRISPR pooled libraries! Addgene’s collection also includes a base editing sensor library, a base editing ...the only game in town! Pooled libraries can be designed for all kinds of different high-throughput experiments...elements using golden gate cloning. This toolkit is designed to be used across many species of bacteria. Surface...Libraries has tips on using these libraries and designing screens with them. All pooled libraries are available...

...have pre-designed plasmids for these systems! Ion sensors One common biosensor class is designed to monitor...all have specific sensors designed for them. For example, this class includes tension monitors associated...interest for many disciplines! Most of the sensors designed for calcium generate a fluorescent output, which... fluorescence in the presence of calcium. This includes FRET-based sensors such as Twitch. While calcium.... Physiological factors Unlike the previously described sensors, this class measures either factors as...to use them, you’ll need to use the vectors (or redesign the tools). It’s a good thing, though! Viral delivery...

...please improve your plasmid maps!" After thoughtful design, vetting, and tweaking, we’re excited to announce...identifies plasmid features that differ by only a few nucleotides, such as the fluorescent proteins EGFP and mEGFP... software. In fact, SnapGene’s feature library includes nearly 3x as many features as could be detected...greater than our previous mapping software and now includes Type IIS restriction enzymes that are commonly...the gene, the name of its gene product, and a description of its function. You can also click one location...enzymes that cut the given sequence. The table includes enzyme names, the locations where the enzymes ...features detected in the plasmid sequence. The table includes feature names, locations, sizes, colors used to...