...keep up on all of the latest protocol videos, lab tips, and more video content from Addgene, subscribe to...viral genome sequencing strategy to confirm the identity and purity of all AAV viral lots. Aligning with...community. Here are a few virus-related protocol videos that may help to save you time and improve your... by iodixanol gradient ultracentrifugation This video goes over AAV vector purification using an iodixanol...critical tool for many scientists. Mammalian cells provide scientists with the means to study biological processes...If you’re new to tissue culture, check out this video for some tips and best practices for getting started...the Addgene blog Learn about Addgene's Lab Tips video series Resources on Addgene.org Find lab protocols...

...imbalance of electrical charge between the inside and the outside of the cell. This imbalance is maintained...Hyperpolarizing chloride channel SwiChr++ - A channelrhodopsin converted into a chloride-conducting anion...Addgene Optogenetics guide Primer on Optogenetics blog series Addgene Biosensors guide Other resources ...alter cell types and patterning There is mounting evidence that signaling via ion channels and even between... also affect patterning and in some cases even override genetically encoded patterning. Based upon trends...Planarian worms cut into three segments “remember” which side should grow a head and a tail, and when gap junction-mediated...encoded fluorescent voltage indicators (GEVI) are ideal tools to track bioelectric states (Matthew et al...

...significantly reduce the risk of an accident (the most common reason for lab accidents is not following safety protocols...article we will discuss general viral safety considerations and best practices for handling lab-grown viruses...replicate and continue the infection cycle once inside the host. While the virus can still enter (and ...and safety department if they are not currently provided. Eye protection or face shields are required ...contamination. As most centrifugation has to be done outside of a biosafety cabinet, you’ll need to take extra...equipment are one of the most common forms of lab accidents, so extra precaution should be used to reduce ...than cell cultures do, and tired researchers are accident-prone researchers. Note that safety policies ...

...selection of guideRNAs. Of particular note, ABE8s reported in this work did not cause genome-wide, guide-independent...human cells (Gaudelli et al., 2020). One of the widely used base editing systems, ABE7.10 (and the starting...catalytically impaired Cas protein (dCas or Cas nickase)  a guide RNA that targets the complex of TadA and dCas to... Gaudelli et al., 2020. Directed evolution identifies adenine base editors with improved editing in...the “x” is the placeholder for the number that identifies the mutation in the evolved TadA domain. Because...ABE8.20-m/d) to evaluate further.  The creation of accidental insertions and deletions (indel) by the catalytically...motif (PAM) made up of 5′-NGG-3′ (“N” can be any nucleotide base, followed by 2 guanines). This limits the...

... E. coli genome-wide inhibition library To expand the tools available for genome-wide studies in E. coli...tools for your research! This time: E. coli genome-wide CRISPR inhibition Covalent tethering of DNA template... Nature Communications Find the E. coli genome-wide CRISPR inhibition library Covalent tethering of...enhance HDR repair using a single-stranded oligodeoxynucleotide HDR template covalently attached to the ...base editor (CBE) with rat APOBEC1 causes genome-wide deamination of RNA cytosines in human cells. Thus...Nme2Cas9 with gRNA cassette Erik Sontheimer’s lab identified a Cas9 protein from Neisseria meningitidis, Nme2Cas9... Nme2Cas9, that recognizes a simple dinucleotide PAM (N4CC), lending to high target site density (average...

... event’s success is evidence that, to share science, all you need is a great idea and a group of people...one-day conference aimed at the general public that provides an accessible introduction to various scientific... engage with the public. The organization runs a wide variety of programs, including fall and spring lecture...science cafes, and Reddit AMAs. Their website also provides a variety of short- and long-form written content...year graduate student at Harvard, who wanted to provide a conference-style experience that was accessible...SITN’s strength in the biomedical sciences and decided to offer a varied format event to keep the audience...improve our communication and organizational skills provide great training for a number of alternative careers...

...post has provided some insight on these differences in viral production and will help you decide which is.... In this post, we will compare two of the most widely used systems, mammalian and insect cells. Whether...cells are also easy to cultivate, fast-growing, and widely available. The viruses Lentiviruses, AAV, and adenoviruses...lentivirus production). The packaging plasmids are widely available, so you’ll only have to worry about cloning..., they are easy to cultivate, fast-growing, and widely available. Unlike mammalian cells, they do not ... (about three to four days), this second round provides the added benefit of producing a stock P1 virus... your viral supernatant. Baculovirus replicates inside insect cells, and its lifecycle ends in cell lysis...

...important factor to consider is your desired library coverage, since this will help decide what transformation...and how will you ensure this?). You should also consider if there are any features of the library backbone...difficult to transform.    Figure 1: Factors to consider when creating a pooled library amplification protocol...amplification protocol is the Chong Zhang E. coli Genome-wide Inhibition Library. DNA and cell amounts After ... work! Selecting your media Lastly, you need to decide between growing your library in liquid media or...similar in size and composition as examples. Table 1 provides an overview of pooled library protocols you can...DOCX, 15 KB) CRISPR Activation Human Genome-wide CRISPRa-v2 Libraries 104,540–209,080 20 bp ...

... tumors, and moreso than other methods, the identification and isolation of rare cell types. In recent...which spectral flow cytometry requires special considerations. Conventional flow cytometry During conventional...flow cytometry, light emitted by a fluorophore is guided to a single detector using a series of mirrors ...Instead of using a single (primary) detector to identify a fluorophore, spectral flow cytometers use all...thought of as a series of histograms flipped onto its side and compressed into a heat map. BV421 peaks in V1...cytometry for a given experiment, it is important to consider how many parameters you want to use in a given...and spectral flow cytometry, it is important to consider the expression levels of the markers you are including...

...here to help with that! Four important factors to consider for any experiment are: Speed Cost Difficulty...For cloning in particular, another important consideration is: The number of inserts ...as some methods...versatile Type IIS restriction enzymes. These cut outside their restriction sites allowing you to easily ...little as 30 minutes! Alternatively, you might consider Gibson cloning. In this technique, you first perform...order determined by their homology. You might also consider FastCloning, first described by Chaokun Li and...Finally, TOPO cloning is another good option to consider when time is of the essence. This method requires...-generated insert. When mixed, topoisomerase I provides both the endonuclease and ligase function while...

... or ddPCR is a method that provides ultrasensitive and absolute nucleotide concentration, unlike qPCR ...withstand changes in temperature. Taq was first identified in the late 1960s during research at hot springs...addition to Taq DNA polymerase, PCR requires free nucleotides (dNTPs), template DNA to amplify from and unique...polymerases require an existing strand of DNA to add nucleotides to.  Using these reagents and a series of heating...temperature is then raised slightly to Taq polymerase’s ideal temperature (70-75oC). At this temperature Taq polymerase...procedure check out our PCR protocol page, protocol video and reference on how to design PCR primers.  Types...PCR is used commonly in disease or pathogen identification. Scientists can simultaneously detect several...

...lab provide expression selectively in Parvalbumin containing cortical interneurons. We now provide ready-to-use...through our hot plasmids articles. These articles provide brief summaries of recent plasmid deposits and ...cell’s surface. The Bloom lab used the library to identify RBD mutations which do not interfere with ACE2....1074/jbc.RA120.01289     Novel protein cage provides simple enzyme protection in yeast bioproduction...enzymes in a reaction pathway, the MPyV system provides a simple, efficient method for protecting a single...- Ampl133 targets S. cerevisiae genes with 6-12 guides per gene where gRNA expression is induced by anhydrous...anhydrous tetracycline. Three new anti-CRISPRs were identified that inhibit the S. aureus Cas9, a Cas protein...

...homologous recombination (HR). Adapted from an image provided by Manuel Gonçalves; made with BioRender. ...vectors As compared to other viral vectors, AdVs can provide a large transgene packaging capacity. For instance...chromosomal region. The CRISPR/Cas9-derived RNA-guided nuclease-induced gene disruption frequencies in...in corrective genes. For this you would need to provide a DNA template to repair the double strand break...Nature Methods the same year that delivering RNA-guided nucleases or TALENs together with AdV donor DNA...demonstrated by Manuel Gonçalves and his colleagues at Leiden University Medical Center are now available at ... Dystrophy, Alpha1-antitrypsin deficiency (most widely used so far) or for cancer gene therapy such as...

.... Amp is a semisynthetic derivative of the more widely known antibiotic, penicillin, and is a beta-lactam...buys you a little more time, since bacteria won’t divide immediately after it has been heat-shocked or electrically... it by producing a lot of beta-lactamase. Pros: Widely available, cost-effective, timesaving for transformations...Expensive.   Kanamycin Kanamycin, or kan, is an aminoglycoside which inhibits protein synthesis. The kan resistance...The NPTII gene confers resistance to several aminoglycosides, including G418 which is commonly used for ...recovery period than amp or carb (60 minutes). Pros: Widely available, cost-effective, confers mammalian resistance... with all bacteria used in research, it isn’t a widely popular selection agent. Pros: Stable (spectinomycin...

... Addgene's Lentiviral Guide Most popular lentiviral plasmids γ-Retrovirus Guide References Cullen, B....retroviruses have been developed into some of the most widely used tools in molecular biology. “Retroviruses”...immune system directly). It may seem odder when you consider that out of the 6 x 106 virus-like particles that...DNA called a “provirus.” This DNA is synthesized inside of what is called the “target cell” (or host cell...reverse transcriptase are packaged (Wu et al., 1997) inside of the capsid . This is useful for viral reproduction... particle, some of the RT protein gets packaged inside of the vector particle, along with its RT-deficient...clock to the 6 o’clock positions at the left-hand side. These steps are common between natural infection...

... of regulation of genome functions, we need to identify molecules associated with specific genomic regions... genomic regions can be easily isolated for identification of associated proteins, RNAs, and other genomic... retroviral vectors expressing tagged dCas9 and guide RNA. The retroviral enChIP system enables us to ...system will allow researchers to use enChIP in a wide variety of biological systems. We are currently ...addition, we have recently developed the oligoribonucleotide (ORN) interference-PCR (ORNi-PCR) method ...shipping. Addgene realizes the One-Stop shopping for a wide range of plasmids. The merit of depositing plasmids...community is a kind of duty for researchers when considering the peer-review system of journals and grant ...

... While not ideal, transient expression of viral constructs is a useful tool as it provides a means to ...the specific target cell or tissue needs to be considered. Q2: Can I use lentiviral transfer vectors for... for the HIV Tat protein and consequently are considered safer. Second-generation transfer plasmids MUST...of lentiviral vectors in producer cell lines. Identifying less toxic envelope proteins or envelope proteins...to produce viral particles. The components are divided as follows: Transfer vectors contain minimal cis-acting...those three genes plus tat. The envelope plasmid provides a heterologous envelope for pseudotyping and is...to lower the risk. Biosafety should always be considered with respect to the specific experiments being...

... let’s say you identified gene target X for BRCA-deficient breast cancer and identified several candidate... of two approaches. The first route involves identifying a gene/pathway to develop a drug for (aka a druggable...to their target, or their direct target can be identified if it isn’t known. If a strong candidate emerges...put into them in those later stages. Target identification Not all diseases are fully characterized or...simple pathology. In these cases, preliminary identification of potential targets by CRISPR can accelerate... activate, or knockout many genes at a time to identify ‘hits’ - genes that affect disease progression...off target effects, both of which make it a non-ideal drug. If a drug selectively kills the wildtype line...

...need to be inside of a gene’s open reading frame, in fact, they can be activated a considerable distance... gene expression are all factors to consider. Once you’ve decided on the type of trap, there are many...world, approximately 50% of predicted genes have unidentified roles as well (Vanni, et al). Is it true that...genes are truly dispensable or are the current identification methods missing something? Here we will review...review some time-tested gene identification systems that may have a leg up on their modern counterparts...just yet. Gene and promoter traps unambiguously identify the gene that they are monitoring as they are ...distance away from a gene which can make identification of the gene itself difficult. The best trap depends ...

...we can confirm the identity of the gene and check for any mutations. Once we identify the gene, we can ...talked about how our new Snapgene generated maps provide improved feature detection with an easy to use ...SnapGene for plasmid quality control are: To identify important plasmid features To align NGS sequencing...experiments for further plasmid verification Identifying plasmid features While our quality control process...in Snapgene’s extensive database. Snapgene also provides easy visualization of open reading frames, which...similar features and it allows you to more quickly identify the same feature in other plasmids. Aligning ...Aligning sequences with Snapgene    Once we’ve identified the features within a plasmid, we next verify that...

...range, identify a few of the features that confer broad host range in plasmids in nature, and provide tips... meliloti, Rhodobacter sphaeroides, R. capsulatus, Rhodopseudomonas spheroides, Salmonella spp., Serratia...equally. Only a handful of plasmids have been identified as broad host range plasmids. The properties ...are not fully understood, but scientists have identified some of the features that confer broad host range...metabolic load (Meyer 2009). Figure 1: Idealized examples of a narrow host range plasmid with only...Porphyromonas gingivalis, Proteus mirabilis, Providencia spp., Pseudomonas spp., Rhizobium leguminosarum... M. xanthus, Proteus rettgeri, P. mirabilis, Providencia stuartii, Pseudomonas spp., Rhizobium leguminosarum...

...Any ∼20 nucleotides of DNA can serve as the target for Cas9’s single guide RNA (sgRNA), provided it meets...unique experiment. Here, we will introduce you to the wide array of Cas9s (many available through Addgene’s...Specificity concerns? If off-target effects are a worry, consider a dual-sgRNA system. This system uses a “nicking...little homology to other regions of the genome and consider using a Cas9 variant with higher specificity (...residue of a protein or knocking out a very small peptide. In these cases, you have little flexibility as...specific? Perhaps you need to introduce a single nucleotide modification at a defined amino acid to mimic...requirements, giving it more flexibility. A consideration with this tool is that the desired edit will...

...accurately communicating ideas in a content-rich, easy-to-understand set of PowerPoint slides. What are some of...learned as a healthcare consultant and give you an idea of what a career in this field involves, and why...a defined path forward. Communication is key as ideas must be relayed quickly both internally and externally..., where case managers are encouraged to use the wider firm’s resources when their teams are facing a crunch...approach brings to patients, doctors, and insurance providers, and is crucial to how our clients’ products will...you’ll have the opportunity to learn the business side of consulting cases, while feeling like you are ...to get your foot in the door When I was first considering preparing for and transitioning to a career in...

...UnLOCKING Cas13 (C2c2) was first identified in 2016 by the Zhang lab as an RNA guided RNase (Abudayyeh et al 2016...-related single nucleotide polymorphisms (SNPs) from human saliva. SHERLOCKv2 provides a method to detect... expensive, time-consuming, and complex to use outside of standard laboratories. In the case of the COVID... 2016). Cas13 can be guided by a single CRISPR RNA (crRNA) to cleave ssRNA or mRNA. It also exhibits a...based on DETECTR technology with the mission to “provide a CRISPR-based platform on which an infinite number...2016) C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 353:aaf5573... Koonin EV, Zhang F (2015) Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell...

...searched for this unique sequence of DNA and both identified lymphocyte antigen 6 complex (LY6A) as a receptor...library of AAV9 mutants that had a small randomized peptide loop inserted in the AAV9 capsid. This mutant library...then screened in vivo in C57BL/6J mice strains to identify variants that reached the CNS following IV injection...hematopoietic stem cells. Its ligand(s) has not been identified. While LY6A or other Ly6 gene family members ...susceptibility. The Deverman and Wilson Labs independently identified LY6A as a receptor for PHP.B by looking for a...total, they tested 13 strains, with seven strains identified as permissive and six as non-permissive to PHP.B...the PHP.B permissive phenotype of mouse strains beside C57BL/6J and BALB/cJ, and the effects of overexpressed...

... understanding of CRISPR, and perhaps provide insights and ideas for your own research! Classification... abundant of the two, comprising about 90% of identified CRISPR-Cas in bacteria and nearly 100% in archaea...transposases, by leaving out Cas3. Type I is further subdivided into subtypes A–G. These subtypes have similar...Schematic of a Cascade complex (blue subunits) with a guide RNA (green and orange) bound to the target DNA with...with BioRender.com.   Type III Type III is considered the most complex of the CRISPR types and is thought...et al., 2021). IV-A and IV-B lack what you may consider to be an integral component of CRISPR — a nuclease...engineered Cas enzyme, with adaptations for increased fidelity, activation/repression, high precision edits, ...

...need for target-specific antibodies and have been widely used across species and applications. In this blog... What are epitope tags? Epitope tags are short peptides introduced at the N or C terminus of a protein...purification or detection of the tagged protein. These peptides consist of as little as 8 or as many as several... (rabbit). Myc The Myc tag is a 10 amino acid peptide (EQKLISEEDL) derived from the protooncogene of ...immunofluorescence . However, protein purification is not ideal with Myc, as low pH conditions are required during...these species. HA The HA tag is a 9 amino acid peptide (YPYDVPDYA) derived from human influenza hemagglutinin... chicken) to this tag! GFP  GFP is more than a peptide – it’s a 238 amino acid protein that can also be...

...chance. Luckily, around the time I decided to look for work outside of academia, a job as a ‘Cancer Marketing...next and what options lie ahead of me? Like a considerable number of researchers, I had high hopes of securing...in the business of western blots and cloning, I decided to pack it in. I had just started month 8 of my...my colleagues and what I was researching, but I decided I needed a new challenge. Looking for sales and...yourself, sales and marketing positions like these are widely available, especially for life science researchers...purchased their products, so you have some of the best ideas on how to sell these products to other scientists...marketing knowledge. For most PhDs looking at careers outside of academia, sales and marketing can be great places...

...self-reflection necessary to identify areas that need work or growth. Here are some ideas for making your meetings...do you make the most of this new relationship? Consider adding formality and active goal setting to your...learned, especially noting action items that are identified. Keep a journal to record topics, plans, findings...you can find it in our Mentoring for Scientists Guide.  You can use it to clarify and discuss values. ...Attainable, Relevant, and Time-bound. It is a good idea to take larger goals (e.g. Figure out what I want...items and make progress. To illustrate this point, consider that I try to exercise every morning, but the ...postdoc can mentor a graduate student – you get the idea. There are many advantages to being a Mentor: Learn...

...promoter and the gRNA scaffold are provided with the kit. Oligonucleotides specifying the gRNA seed sequence...promoters. To use this plasmid, you simply order oligonucleotides for your chosen gRNA target sequences and ...uses Type IIS restriction enzymes, which cleave outside of their recognition sequence, creating flanking...Cas9 Golden Gate multiplexing is to clone the oligonucleotides specifying each gRNA target sequence into ...rectangles) are cloned into various plasmids using oligonucleotides. These plasmids contain Type IIS restriction...allows you to express 2-4 gRNAs, with four being the ideal number. First you generate four unique kanamycin-resistant...you to express two gRNAs constructed using oligonucleotide assembly.  Unlike some of the plasmids described...

...the gRNA (composed of a ~20 nucleotide targeting sequence and ~75 nucleotide scaffold sequence) is consistent... storm owing to its ease of use and utility in a wide variety of organisms.  While much of current CRISPR...being realized. There are a variety of reasons to consider using genome editing to change the genetic code...developed in other model organisms (see our CRISPR guide for common CRISPR principles). However, plant-specific...hereafter referred to as Cas9) and a single stranded guide RNA (gRNA) is sufficient to modify the genome of...nanostructures (Zhang et al., 2019) or cell-penetrating peptides (Santana et al., 2020) and plants viruses (barley...deliver CRISPR-Cas9 to plant cells and should be considered alternative to PEG or agrobacterium-mediated ...

...interaction allows scientists to easily purify and identify any proteins that are tagged with biotin using...biotin-phenoxyl radical in the presence of hydrogen peroxide. This radical reacts with electron-rich amino ...hours like BioID. APEX has allowed scientists to identify the proteomic map of the human mitochondrial matrix...multiple rounds of directed evolution until they identified a mutant with significantly increased sensitivity... binary protein-protein interaction as well as identify additional interacting proteins in one simple ...uses proximity protein labeling based on BioID to identify RNA-protein interactions (Ramanathan et al., 2018...monoglycine residue at the N terminus with a small peptide (LPETG). In EXCELL, the mgSrtA variant can be displayed...

... you can feel lost. Use this post to explore the wide range of careers available to scientists and open...from Grad Student to Entrepreneur Science careers divided by the most pertinent skills required Research ...and carry out in-depth research is applicable to a wide range of industries and is a highly desirable quality...including: Science Reporter Scientific Sales Epidemiologist Scientific Recruitment Meteorologist Research...explore the different options available and then decide on a course of action. Instead of rushing in, you...into a career, do your research Once you have identified possible careers, the next step is to research...computer programming skills? Other things to consider are wage, opportunity to advance, and how many...

...experimental needs, and can transduce (infect) a wide range of cell types with a high efficiency. However... part of the host genome that can replicate and divide along with the endogenous DNA. AAV also has low...AAV is also a cool tool in the laboratory! It is ideal for whole animal administration, such as in mouse...alternative virus, or other strategies may need to be considered if size constraints are an issue. Adenovirus...hamsters, although no cancer-connection has been identified in humans to date. Over fifty serotypes of human-infecting...human-infecting adenovirus have been identified so far! Adenovirus generally has high immunogenicity, ...viruses used in biological research, you might have an idea which ones could be helpful to you. Interested in...

.... Manufacturers rarely provide the construction plans or CAD (computer aided design) drawings of their...users the chance to understand the "black box" provided by the manufacturers and enables customized experiments...limitations into account in the development process. Widely used, commercially available tools such as the ... the community to regularly replicate designs worldwide. Distributing hardware via globally established...MIT), and documentation (e.g. CC-BY) allow users outside the project to modify the raw and design files,...of work from universities and research centers provides a potential conflict between traditional IP-based... for innovative organizations and companies to provide problem-solving platforms.     Fig. 3: ...

... between these methods is that both attempt to identify new players in a biological process. Players can...approaches can be found. As an example, say you are considering either a cellular screen or a selection. A selection...molecules/drugs. With the advent of CRISPR, genome-wide knockout screens are possible with gRNA libraries...specific to your scientific question. Things to consider when selecting an assay system include: How specific...t yield any hits. Make sure you plan ahead and consider these questions to save yourself a headache later...population. For these experiments, the goal is to identify manipulations which either enhance or suppress...scene, there are other options which are worth considering. RNAi screens are similar to CRISPR, but instead...

...” has me considering the microbiome more often than not). If you’re someone who’s considering getting ...definitely enjoyed the lab!) and would therefore consider myself a neuroscience novice (newb). In this post...entire story-telling session organized by Story Collider and which featured a talk about the different ...Developing better AI programs gives neurobiologists new ideas about how the brain functions and vice versa. Even...getting into neuroscience from the mol bio side you’re sure to have many opportunities to learn about and develop...large data sets. Whether these data sets include videos of behavior, many many images of brain regions,...particularly interested in using AAVs because they provide a highly versatile means of delivering genes to...

...’s social media toolbox. Besides advice for using Twitter, this post provides suggestions for using LinkedIn...Now I write for the @Addgene blog & I'm my lab's resident #scicomm expert. What tips do you have for using...discuss how scientists can start using Twitter and provide real-life examples of how they and others have ... February 13, 2014 2. "The Scientist's Top 10 Guide to Twitter" blog post from Dr. Paul Knoepfler Hopefully...pknoepfle 3. "Social Media as a Scientist: A Very Quick Guide" from Nature Jobs Blog While Twitter is a popular...sheet from Science Media Savvy This tip sheet provides advice for tweeting and blogging as a scientist...Addgene blog. 5. "Social Media in Your Working Day" video from Science Media Savvy At this point, you might...

...team of twenty excited undergrads. Team IISER-B’s idea for the iGEM 2019 is to take bacteria out of their...objective is to induce tolerance towards cold, we decided to focus on a special class of enzymes expressed...usually defined between a fairly nippy 20°C to a decidedly uncomfortable 45°C). Their more adventurous cousins...fruit juice clarification, meat tendering, and oligosaccharide synthesis. A major incentive to use cold-active...industry as it can potentially help minimize the incidental losses like spoilage and improve the productive...inefficiently that no industrial operation would ever consider them worth the trouble.  Enter ELSA: E. coli Learning...Acclimatisation To resolve this impasse, our team decided to create a hybrid system where we could incorporate...

...maintenance within an E. coli cell, but vectors can be widely utilized across many different cell types and each...budding. Yeast Centromere plasmids (YCp): These are considered low copy vectors and incorporate part of an ARS...are most similar to bacterial plasmids and are considered “high copy”. A fragment from the 2 micron circle...most commonly used selection markers in yeast and provides the element needed to overcome the auxotrophy ...as additional uses for said element. This link provides a more extensive list of yeast auxotrophic markers...auxotrophic selection in E. coli? Additional considerations HIS3 L-hisitidine no S. cerevisiae  no yes...experiments. MET15 L-methionine and overproduces hydrosulfide ions yes - Grow with methylmercury. S. cerevisiae...

... approaches a host cell surface are complex polysaccharide structures, many of which are easily varied...proteins could function as surrogate antibodies for identifying and characterizing sugars and sugar-containing... anything else, making it difficult to quickly identify possible gene function. In this post, I will describe...assay, including examples of RBPs successfully identified using this method. Step 1: Prepare a random fragment...transformants for appropriately sized inserts. We consider a library worth using if at least 40% of colonies...overnight), flip the membrane over and place it colony-side up onto a fresh LB plate containing an appropriate...onto a lysis solution-soaked filter paper (colony-side up) and leave for 1 h at room temperature. After...

...missense variants identified from 58 individuals with GNB1 disorder since the identification of this disease...and Accelerate Research Discoveries) Challenge provides grants for rare disease research. This year is...other symptoms as discussed in this educational video. “SLC6A8 belongs to a large family of solute channels...perform a functional screen in mammalian cells to identify any solute channels that can compensate for the.... “This proof of concept study will help in identification of new targets that could greatly enhance our...keratinocytes,” says Mellett. This would help the lab identify therapeutic targets in the future. Congratulations...will express these candidate genes in TS iPSCs to identify whether any rescue cell migration and contraction...

...Instead they are detected with tyramide signal amplification. Tyramide signal amplification occurs when...are secreted from cells due to their IgGκ signal peptide sequence, making them easy to harvest from cell...when HRP converts a fluorescently labeled tyramide molecule into a highly reactive radical, which is then...Detection Method Uses Pros Cons Antigens HRP (P) Tyramide signal amplification of HRP Staining cultured...Enzymatic reactions on tissues can be cumbersome Tyramide reagents can be expensive Can only stain with ...of a nanobody that would make a useful RANbody, consider leaving a link to its sequence in the comments...immunodetection If you enjoy DIY lab projects, consider making you own DNA ladders with these plasmids...

...enzyme-bound hydropersulfide that is then transferred to thiol-containing acceptors. These hydropersulfides modulate...live cells. His work focuses on the enzyme hydropersulfide-propagating enzymes 3-mercaptopyruvate sulfurtransferase... susceptible to redox modification, the MPST hydrosulfide can be transferred to the roGFP2 cysteine thiols... to better understand the processes that happen inside the cell,” says Pedre Pérez. Figure 2: ...she studies opioid use disorder. Her goal is to identify cell populations in the brain that contribute ...neurons at specific firing frequencies. She hopes to identify how in vivo firing frequency of PV neurons affect...genetically encoded voltage indicators. Tian developed a video-based high-throughput screen for directed evolution...

... 1 we provide an example of such a comparison between cyan fluorescent proteins using widefield imaging... which usually are undesired properties. In the ideal situation, a fluorescent proteis should emit a stable...since different fluorescence imaging strategies use widely varying amounts of excitation power (Shaner et ... intense light (for a brief time), whereas in widefield imaging fluorophores are excited with relatively...measurements and hope this information can help guide your understanding of reported data. Photobleaching...Measuring photostability at realistic powers will provide better insight into the photostability in the intended...situation. Measuring photostability in living cells provides a much more realistic view of photostability. ...

...want to reconsider your approach, and try a different cloning strategy. Once you've identified potential...plasmid and all the steps you will have to take. Decide which technique you are going to adopt (i.e. restriction...digestion on an agarose gel. If the digestion does not provide the fragments of the size you expect, check the...region of the primer to 40-60 bp. It is always a good idea to perform primer optimization, especially if you...that, they can be open source (i.e. primer3) or provided by the major companies selling PCR reagents. Nowadays...Now that you've read our plasmid troubleshooting guide, you hopefully have some new strategies to tackle... the lab he develops new assays and processes. Outside the lab, he enjoy the outdoor in all the possible...

...secondary considerations to validation, application specificity, and experimental considerations - no other...you know which ones to try?  Read on and we will provide you with some advice on the antibody selection ...antibody first look at the application-specific data provided on the manufacturer’s website. Because of how ...has been validated in your specific application. Consider also that there may be variations within an application...section then a directly-conjugated antibody will provide better resolution as these immune complexes are...abundance on a Western blot, a secondary antibody is ideal as it will help to amplify the signal. Some applications...the species that the antibody cross-reacts with. Ideally you will find an antibody that has been validated...

...-art methods of sex and gender analysis and 2) provides case studies with concrete examples of how gender...capacity of stem cells. Yet, very few researchers consider the sex of their cells when doing experiments—...between male and female cells. The result of not considering the sex of the stem cells was that their male... in Norway, the team realized they should also consider the sex of the stem cells. In this case, they ... many researchers I’ve talked to who’ve never considered the sex of their cells. For one thing, cells ...hard to determine. To remedy this, Shah et al. provide a table indicating the sex of the most commonly... there are numerous methodological articles to consider—we have loaded those we are aware of in our checklist...

...just isn’t always ideal. But the increased flexibility helps even when it’s not an ideal site: moving several... that you hope is present next to the "perfect" guide sequence for your genome engineering experiment....editing landscape. The need for PAM-less editing   Guides for CRISPR are typically selected by the ‘quality...sequence, that of SpCas9, is NGG. Assuming all nucleotides are represented equally, that’s only a 1/16 chance...flexibility in gRNA selection and subsequent editing. Identifying PAM-free and -flexible Cas enzymes   The solution...more pan-genome PAM flexibility, you’ll want to consider one of several Cas9s that recognize NG PAMs. The...sought to address the PAM problem through structure-guided engineering of the PAM-interacting domain of Cas9...

...trials involve identifying a therapeutic gene, finding a means to deliver it and identifying a suitable route...technologies have allowed scientists to quickly identify underlying genetic causes of some human conditions...example, it was possible to transduce the T-cells outside the body and thereby correct the mutations using...productive infection. AAV vectors are engineered to provide additional safety benefits: they lack all viral...immunity. AAV vectors are also capable of infecting a wide range of cell types including but not limited to...through direct evolution, capsid shuffling, and peptide displays. These capsids will be selected both for...to other gene delivery tools that are under consideration, recent advancements in various AAV technologies...