...making the purification process simpler. Due to its high yields, E. coli is often used for the production...transfection, depending on whether the heavy and light chain genes are contained to one plasmid or if they...on an SDS-PAGE gel. Once stained, the heavy and light chains become visible alongside your ladder and ...software. Any bands on the gel beside the heavy and light chains can also be measured. Contaminating bands...Rockberg J, Chotteau V. Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production...capture process based on magnetic beads from very high cell density suspension. Biotechnol Bioeng. 2021...

... information about insert orientation, then you might consider designing orientation-specific primers....primers. Blunt end cloning is an example of when you might want to know the orientation of the insert. One member...with this and a few other colony PCR tips are highlighted below. Preparing template: Pick a single colony... liquid cultures. Lastly, you can start small overnight liquid cultures with the clones you pick and only...size. Make sure to use an appropriate molecular weight standard for reference and to add a loading dye...

...James from Binning Singletons. During Junior High and High school in the 80s and 90s, Joe was that kid ...asked if she had a few minutes to point me in the right direction for my future work. She graciously took...match people within similar fields (ASM Microbe has eight tracks), something we didn’t have enough people ... ASM Microbe, we connected people who otherwise might never have met. Binningsingletons.com will be the... but before going to graduate school, Joe taught high school chemistry for a year. The teaching/mentoring...

... advice on areas where you might need some help. Today, we’re highlighting an oft-underlooked transferable...skate as a starting point, their ideas scored 37% higher in originality. It may not be easy to optimize ...truly disagrees with the group (because they will fight more adamantly for their solution), selecting a ...power to change anything. You may not have the highest position or the longest tenure, but don’t let that...

... confirmed that indeed the gRNA-pMB1 had a slightly higher leaky expression in BL21(DE3) that resulted...customizable cargo gene in the form of RE-cargo-LE (RE, right end; LE, left end) pTnsABC expresses the transposases...glucose dehydrogenase expression that is 2.6 times higher when compared to the industrial strain. In our ...copies of integrated genes. We obtained a stable and high-yielding strain which had 6 times the yield of the...transfer on transposition-inducing agar lead to higher cargo copy numbers. (D) The effect of gene dosage...

...Genome-wide CRISPR/Cas9 screens are a high-throughput systematic approach for identifying genes involved...alternative to genome-wide RNAi screens, which although highly effective, are affected by low on-target efficacy...unclear if off-target cuts confound the results of high-throughput CRISPR screens. Do short guides (17-...mismatches more distal to the PAM, and 2) guides with high GC content have greater off-target activity. Short... with only a small loss of on-target activity in high-throughput screens. The human and mouse versions...measurement of off-target activity using Cas9 toxicity in high-throughput screens." Nature communications 8 (2017...

...our plasmid lab to develop a reliable and robust high quality viral vector service, making an array of...protocols we use in our lab — being able to order high quality viral vectors from the Addgene repository...on quickly in the scientific community, turning slightly under ten requests a day in 2017 to over fifty...that are activated, respectively, by substrates, light, or engineered ligands (Figure 2). Biosensors, which...our own repository data, giving us significant insight into which vectors are likely to be of most use...plasmid repository gave us the experience to launch a high-quality ready-to-use viral vector service. But in...

...chance of cross-reactivity Sandwich High specificityHigh sensitivity Longer protocolIncreased chance...several drawbacks. Direct ELISAs generally have higher levels of background staining. Antigen-containing...simple protocolReduced chance of cross-reactivity Higher level of background stainingLess sensitivityLess...availability of conjugated antibodies Indirect High sensitivityGood antibody availability Longer protocolLess...Antibodies 101: ELISAsAntibodies 101: Selecting the Right Antibody Antibodies 101: Polyclonal Antibodies Resources...

...which cuts at NRN PAMs with high efficiency, as well as NYN, but with a slightly lower efficiency. Information...available, it can be difficult to decipher which one is right for your unique experiment. Here, we will introduce...the genome and consider using a Cas9 variant with higher specificity (eSpCas9 and HypaCas9) instead.  Choosing...location and type of edit you need to introduce highly specific? Perhaps you need to introduce a single...choose from within the Cas family if Cas9 isn’t right for your experiment. Keep an eye out for our upcoming...

...up-regulated. For instance, generally, the more highly expressed a gene is under native conditions, the... achieve using a CRISPRa. Your gene of interest might already be hitting an upper bound of activation ...scale CRISPR-based screens can be used to gain insight into protein structure and function (Pan et al...., Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient Cas9-mediated transcriptional programming...doi.org/10.1016/j.cell.2014.09.029 Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena ...j.cels.2018.04.011 Polstein LR, Gersbach CA (2015) A light-inducible CRISPR-Cas9 system for control of endogenous...

...Pitfalls of Fluorescent Protein Fusions Observing a bright signal for the first time after cloning an FP fusion...studies and those of others (5, 6, 7, 10, 11) highlight the very real consequences of misfolded dark FP... blue or cyan and yellow moxFPs. The inherent brightness and spectral characteristics of the moxFPs are...standard FPs. The increase in signal is not due to a brighter FP, but rather because of the correct folding ...listed above. In addition, because the moxFPs are highly monomeric, they are an excellent choice for fusions...

...reported in PLOS publications. The policy was not an overnight revelation, rather it was the result of consultations...A distinct (perhaps similarly impoverished lab) might apply different or novel tools and assumptions to...It could be argued that data generators may, in light of the policy, hold onto data longer before publishing...include suggests pragmatic adoption (although it might be argued various authors have simply chosen to ...significant effort. Our standards of data recording are highly variable and usually left to individual researchers...contact more senior colleagues. Secondly, science is highly technical and this has a natural, albeit unintentional...

...Immunofluorescence (IF), is an immunoassay that brings to light the cellular world. The technique allows you to ...indicates your target’s location. Some other terms you might see associated with IF are immunocytochemistry (... of other antibodies. But, once you do find the right protocol, multiplex IF can give you impactful and...sample on slides, and confocal   Figure 2: High-level overview of an indirect immunofluorescence...ones? Choosing a secondary is often much more straightforward than choosing a primary, so we’ll start there...the future.  We hope that this post has shed some light on immunofluorescence. The technique can provide...

...kDa single polypeptides that contain the variable light chain (VL) and variable heavy chain (VH) of an antibody...minimizing oligomerization of the scFv. While you might assume that the variable domains of an scFv would...lower affinities, lower long-term stability, and a higher likelihood to aggregate due to their small size...library with plate-bound antigen helps select for high affinity scFvs. Bound phage are then proliferated...with plate-bound antigen to select for scFvs with high affinity binding. Bound phage are then proliferated... since direction injection to the eye results in high drug concentrations in the eye with minimal systemic...

...contract citizens enter into in order to protect their rights (2). Less formally, governments are the organizations...public will lose trust in scientists if the march highlights data supporting unwanted results (14). Some loss...surrounding a matter or policy should have more weight than how any one person feels about it. And yet...evidence’ on Trump’s wiretap claim The Chronicle of Higher Education: “March for Science” Organizer Says It...Science Foundation: Universities Report Fourth Straight Year of Declining Federal R&D Funding in FY...

...Every few months we highlight some of the new plasmids, antibodies, and viral preps in our repository...microscopy (cryo-EM) protocols typically require high concentration and purity of the target molecule,...or hundreds of particles surrounding each of the highly-visible beads on the cryo-EM grid.     ...denaturated (left) or complexes may dissociate  (right). B) Protein samples protected from AWI by helical...authors to determine structures at comparable or higher resolution than using more complex protective methods...

...heterodimers composed of one of 18 alpha and one of eight beta subunits. Both subunits have short intracellular...ectodomains can extend and open, binding ligands with high affinity. This extended-open state is key to strengthening...flexibility is crucial for their function. With the right research reagents, researchers can measure this ...developed and defined. According to the Springer lab, a highly specific function-blocking antibody, αVβ3.13, may...as new reproducible rat or mouse rAbs. The other eight are chimeric antibodies composed of rat or mouse... recently showed RGD-binding integrins demanded higher tensions before they would enable cells to spread...

...their regenerative abilities is expected to shed light on human aging pathological developments (Vieira...amphibians in science with early research providing insight into some of the most significant and fundamental... Image from Holger Krisp. The frog genome has high synteny with that of humans so they are often used...the world. With its extensive scientific history, highly characterized genome, and widespread utilization...produces many seeds), self fertilizing, a small yet highly characterized genome, and is easily maintained ...Genome sequence of the Brown Norway rat yields insights into mammalian evolution. Nature 428, 493–521 ...

... To better highlight the great content contributed by our bloggers each and every month, we've decided...Cpf1 Update: Comparison to Cas9 and NgAgo As you might expect given the excitement surrounding anything...month is from guest blogger James D. Fessenden at Brigham and Women's Hospital. This post describes how to... our posts if you have any questions or useful insights. If you're interested in blogging for us, fill...

...vectors containing high numbers of gRNAs in one overnight reaction. STAgR is highly customizable as vector...In this quarterly blog series, we’ll highlight a few of the new CRISPR plasmids available at Addgene....

...RNA is highly desirable for extending the complexity of genetic circuits, allowing for tighter spatio-...Illinois at Urbana-Champaign. The PUF proteins include eight modular repeats, each of which binds a consecutive..., I think, are endless.” Abil says the new tool might even find therapeutic application in addition to...she recommends using standard BsaI in place of the high fidelity version (despite what the original paper...

...fortunate that there’s a guide that directs cleavage right before the stop codon!  Figure 3: How to ...guide sequences (arrows) and their PAM (bold highlighted text) and cleavage sites (slashes) are shown....plasmid options for templates - see the example at right using blue flame plasmid pmScarlet_C1! After creating...primers to add T7 promoter-left homology arm and right homology arm to the desired insert (e.g., mScarlet...the main drawback of i-GONAD is that it requires a high-concentration, column-purified DNA template.   ...

...fixed sample. Depending on the sample type, this might be frozen or formalin-fixed, paraffin-embedded tissue...Depending on your needs, your sample, and targets, you might decide to do simultaneous IF instead (Figure 1B).... Depending on the goal of your experiment, you might need to quantify the intensity of the different ...spatial relationship between the targets. Or, it might be sufficient to describe or classify the labeled...label a sample that expresses your target(s) at a high level, either naturally or by overexpression, for...similar between the two experiments. If not, it might be that the other antibodies are disrupting the ...

...not having any confounding mutations, the risk is high. You are generating a gene therapy product with ...clinical trials. An off-target rate of 5% is far too high when considering human patients and the diseases...measurements at those sites to get an idea of how high the off-target frequency is (more on this below)...sequencing methods. Candidate site sequencing Sites with high sequence similarity to the gRNA can be sequenced...a DNA mutation, off-targets must be quantified slightly differently. For a comprehensive approach, RNA-seq...library screens, the same principle holds true - the higher the number of distinct editing events, the lower...

...connection to the hiring manager. They might say yes and they might say no but do not confuse networking...scientist in the academic system has many pluses. I delighted in my graduate school years for allowing me to...personal questions can elicit the most valuable insights you will get from the interaction. Most people...great way to practice for real interviews where you might spend a whole day having to talk with strangers....

... all night was hand-picked (like our plasmid samples!) by Addgenies. If you were there, you might have...plenty of delicious desserts. We enjoyed a video highlighting the changes at Addgene over the last 10 years...Perhaps the most heartfelt portion of the entire night was when Melina and Ken called their parents up ...

...customized to highlight different skills and to get the important information in the right place on the...organization is small, any person at the company might be able to help you. If the organization is large...additioanal advice for the science career search.  You might also enjoy the other posts in the Addgene Blog Scientist...

...and can exhibit recombination levels as high as 1-3%. In light of this finding, we now include guidance...leaky expression. On the right non-homologous scrambled recombination sights lead to reduced reversion...strategy should work, these vectors suffered from high levels of spurious expression.  As such, researchers...weak and may require longer waiting periods and/or higher injection titers to achieve adequate expression...

... homozygous knock-in animals can be obtained in eight days. The only animals on a plate, other than the... delivery, screening, and more. Here are some highlights from the past few years:  Repair template design...improvements from the Mello lab resulted in strikingly high editing efficiencies, obtaining as many as 100 independent...corresponding to the same strand as the PAM produced a higher editing efficiency, while for edits 3’ to the PAM...templates. They found that Cas9 RNPs could be toxic at high concentrations, and editing efficiency improved ...

...proximity to an endogenous gene. Each trap uses a slightly different approach (described in detail below)...for the endogenous gene – its expression levels (high/low) and patterns (tissue/timing) can be monitored...screened. Depending on the marker, traps can be used as high-throughput screens to introduce reporters throughout...the top trap – they don’t require insertion in a highly specific gene region and they don’t disrupt the...Resources References Wood, V., et al., Hidden in plain sight: what remains to be discovered in the eukaryotic...