...Densmore’s lab at Boston University have greatly simplified the process of generating multiple E. coli gene...GMAP - A new assembly platform for generating both simple and complex DNA constructs Have you ever wished...Sanger sequencing, respectively. The speed and simplicity of GMAP allows scientists to easily design, generate... tend to show their limits when it comes to simultaneously modifying multiple loci (multiplexing). Currently...(MAGE) (1). MAGE uses recombineering (2) to simultaneously incorporate multiple single-strand DNA (ssDNA...in a broad range of strains. Thus, pORTMAGE simultaneously allows genome editing and mutant library generation...genome editing in multiple organisms thanks to its simple, easily modifiable RNA-guided targeting mechanism...

...They are classic parametric tests and one of the simplest statistical tests available. Tailed t-tests If...groups (two samples and a control, for example) simultaneously, with up to two independent variables. This...that matter. However, this is oftentimes an oversimplification. If your independent variables are affecting... you only look at 2 or 3 pairwise comparisons. Similar to family-wise error, is the false discovery rate...positives/negatives, but they all generally yield a similar result and are appropriate to use if all sample...with Ordinal Dependent Variables: A Monte Carlo Simulation Study. Front. Appl. Math. Stat. Volume 2 (2016...

... by a similar function. After analysis, CRISPR classifications were placed into a fairly simple hierarchy...classification system relies on a combination of sequence similarity, phylogenetic analysis, neighborhood analysis... the same class and type don’t share sequence similarity but are grouped due to mechanisms of action and... ensure every unit of the complex expresses at similar levels. It was only recently that researchers began...subdivided into subtypes A–G. These subtypes have similar compositions and mechanisms, with the biggest differences...

...antibodies are less likely to cross-react with other, similar proteins due to recognizing a single epitope of...monoclonal antibodies here. Recombinant antibodies Similar to monoclonal antibodies, recombinant antibodies...internal control for Western blots, shows that a similar concentration of cell lysate was added to each ...   Enzyme-linked immunosorbent assay (ELISA) Similarly, ELISA is used to detect the presence of a single...expression of multiple proteins on the same cell simultaneously as well as compare between samples. Learn more...localization within the cell or tissue as well. Similar to flow cytometry, you’ll use antibodies to stain...

... Many thanks to our guest blogger Derek Simon! Derek P. Simon received his PhD in Cellular and Molecular...composer. View his blog drsimonsaysscience.org or follow him on Twitter @derekpsimonphd.    References 1. Tye...This post was contributed by guest blogger Derek Simon. Optogenetics is spreading through the neuroscience...generation. Archaerhodopsin (Arch) is activated at a similar spectrum as HR and also results in membrane hyperpolarization...

... abundance of thousands of protein variants simultaneously  With increasing sequencing power, scientists...the effects of sequence variants on abundance simultaneously called VAMP-seq, for Variant Abundance by Massively...the a bacterial periplasmic binding protein, one similar to the protein which gave rise to the iGluSnFR ...artificial leaf replica means creating a “leaf” with similar permeability of water and nutrients and colonization... to a second population. Last, Bernach will simultaneously monitor cell cycle and growth rate using TIMER...

...endless ways and everyone finds their own. Here, I simply outline what works for me, and I hope that it is...will keep you from repeating your work or making similar mistakes in the future. Stay organized Set up...experiments can be found in each folder. This can be a simple .txt document. Record the name, date, and location...track of your exact changes. Alternatively, you can simply make a note in your methods section about the edit...result was, so try not to do this. These are the simple rules that I try to follow in my own research. ...

...gRNA are not to scale.   Let’s start with the simplest multiplexing situation: you only need to express... and two U6 promoters. To use this plasmid, you simply order oligonucleotides for your chosen gRNA target...Transcript Multiplexing Plasmids: These plasmids are similar to the Csy4 polycistronic system described above...assembly, and up to 8 gRNAs may be expressed simultaneously. Vectors for both transient expression and ...small insertions. Multiplexing of two targets simultaneously is possible with efficiency >70%. CRMAGE is... delivered on a different plasmid and allows simultaneous multiplex editing of six different genes. This...

... to provide that context yourself. Simple words are not so simple Just as our lab used jargon that required...problematic word. How many different things do you simply refer to as a “tube”? We took steps towards solving... that nearly all Addgene teams used that seemed simple, but they could in fact be interpreted in several...when starting rotations or my postdoc would have simplified the first weeks or months when I was learning...

...these mutants are not toxic to HEK cells and that similar mutations can improve the specificity of Cas9 derived...from S. aureus. This means you can likely apply similar rational mutations to your Cas9 homolog of choice...SpCas9-HF1 Kleinstiver et al and the Joung Lab similarly thought that if they could weaken sequence independent...found in eSpCas9, the effect on specificity was similar; SpCas9-HF1 generated fewer off-target cuts when...variant displays comparable on-target activity and similar or decreased off-target activity across the sites...

...computational methods to identify genes by their similarities to genes in other species, but these studies...gene function wide open. What if two seemingly similar proteins have acquired divergent functions due ...strains are available. Then, staff or students clone similar sequences from a less-well studied organism into...confident that the gene from the test species has a similar function to the gene in the reference species. ...Out Our Molecular Biology Reference Pages Learn Simple Lab Techniques in Our Protocol Videos ...

...through homologous recombination. The concept is simple: deliver a template with a modified version of ...nucleotide sequences are exchanged between two similar or identical molecules of DNA. Gene targeting takes...insert GFP into the genome, we’d create a plasmid similar to the one shown below, where the sequence of GFP...plasmids described in the preceding sections are simple ways of knocking out a non-essential gene of interest...functions normally because one of its exons has simply been replaced with the same sequence flanked by...

...and a low similarity index. In figure 3, we can see that PerCP and PE/Cy5 have a similarity index of 0.87...Cytek’s website has a convenient tool, called the similarity index, which allows the user to compare the emission...when designing a panel.     Figure 3: Similarity index across 12 different colors on a three laser.... PE/Cy7 and BB515, on the other hand, have a similarity index of 0, which would be a better choice for...

...Charles Gersbach’s lab at Duke University developed similar systems based on the light-inducible heterodimerizing...effector (LACE) system, the Gersbach lab utilized a similar strategy to develop an optimized photoactivatable...system was able to recognize the same PAM and had similar targeting specificity as full-length Cas9 (flCas9...Systems.  Listen to the anti-CRISPR podcast segment Simply expressing anti-CRISPR proteins does not provide...that post-UV exposure it showed cleavage activity similar to the wild-type Cas9. This photocaging technique...Posts Read Our Primer on Optogenetics by Derek P. Simon Read Our Interview with Ed Boyden Resources on ...

...than following it. The Cpf1 crRNA is also much simpler in structure than Cas9’s; only a short stem-loop...found two that display robust cleavage activity similar to that of Cas9. These two nucleases, AsCpf1 and...respectively), both cleave in a staggered pattern similar to FnCpf1. Potential advantages of Cpf1 over Cas9...CRISPR systems based on Cas9 were thought to be the simplest CRISPR systems and the easiest to adapt to genome...et al. suggest that AsCpf1’s off-target rate is similar to that of high fidelity Cas9s eSpCas9 and SpCas9...

...2012). This concept is a little more complex than simply competing for replication control factors and tends... daughter cell during cell division. They don’t simply rely on chance (or random diffusion) to make this...plasmids, incompatibility due to partitioning is similar to incompatibility due to having the same replication...some types low copy plasmids during cell division simply does not allow for enough time for correct partitioning... of bacterial incompatibility groups based on similarity of replication and partitioning systems where...

..., which is not well protected by Cas9. This is similar to the proposed mechanism for engineered pegRNAs...related voltage indicator Ace2N-mNeon also led to similar improvements in performance.      Figure...10.1016/j.neuron.2023.03.009. PMID: 37015225.  Simple and highly specific targeting of microglia with...combination with miR124T targeting sequences, provides a simple yet valuable tool for microglia research and broader...AAV plasmids here! Serrano, C., et al. (2023). Simple and Highly Specific Targeting of Resident Microglia...

... can be used to generate multiple mutations simultaneously (Jiang et al. 2013). Genome editing tools for...small insertions. Multiplexing of two targets simultaneously is possible with efficiency >70%. CRMAGE is...et al., 2021). This pEcCas/pEcgRNA system works similar to the original pCas/pTargetF but has many other...pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays a higher editing...recombinogenic than E. coli, so this system functions more similarly to CRISPR/Cas9 systems adapted for eukaryotes ...knockdown expression of multiple target transcripts simultaneously. Marraffini lab's CRISPRi/a plasmids In addition...

...that you need. The Golden GATEway cloning kit simplifies the cloning process for complex DNA constructs...Grusch et al. demonstrate that Opto-RTKs have a similar level of background activity and activation as ...integrating DNA sequences either sequentially or simultaneously. This method, called by the authors the “Clonetegration...terminator). Next, up to six Level 1 modules can be similarly assembled into a Level 2 vector, thus forming ...repeat-variable di-residue (RVD) module plasmids and simultaneously increases the success rate of module assembly...in place of Brainbow’s Cre/loxP, allowing for simultaneous use of the two systems in different tissues ...the same transgene as the Brainbow cassette, simplifying experiments when additional cross-breeding steps...

...context, they should be expressed at similar levels and under similar conditions as their wild-type counterparts... the fluorophore is pH-dependent. Signal is simultaneously acquired from the the fluorescent and luminescent...now take any open reading frame (ORF) and with a simple recombination reaction, generate a protein with...(MuLE) system from the laboratory of Ian Frew simplifies the process of creating complex polycistronic...tamoxifen-inducible CreERT2 gene deletion, (3) simultaneous expression of cDNAs and shRNAs with a reporter...imaging of multiple endogenous genomic loci simultaneously in live cells. This system allows the study...mCherry), the authors were able to visualize simultaneously several parts of the genome and to determine...

...250 kDa Similar in size 8% 20–250 kDa Similar in size 10% 10–100 kDa Similar in size ...conjugated to primary antibodies). Direct westerns are simpler and faster than indirect ones and can be used in...visualize proteins on a PVDF membrane without staining; simply wet your membrane with 20% methanol and place it...proprietary systems are fast (often <10 minutes) and simple, utilizing individually packaged membrane ‘sandwiches...

...generation simpler, as described in an earlier post on generating CRISPR mouse models. For simple knockout.... It’s also made targeted insertions relatively simple in C. elegans and bacteria. But CRISPRing typical...microinjection, the efficiency of Easi-CRISPR for simple knock-ins and floxed models varies from 8.5-100%...Easi-CRISPR have also recently developed i-GONAD as a simple method for delivery of genome editing components...

...molecules in molecular biology labs (for a review, see Simpson et al., 2015). Visit Addgene's MIcrobiology Pages...following is a description of a technique published by Simpson, Sacher, and Szymanski in January 2016 in Viruses...to remove unbound cells. To detect bound cells, simply lay each membrane on an agar plate selective for...27536293. PubMed Central PMCID: PMC4971087. 3. Simpson, David J., Jessica C. Sacher, and Christine M. ...Chemical Reviews (2016). PubMed PMID: 27319741. 3. Simpson, David J., Jessica C. Sacher, and Christine M. ...

... With this technique, multiple genes can be simultaneously silenced if a consensus sequence is used (~...micro RNAs (amiRNAs) are frequently used. Although similar, amiRNAs are engineered from endogenous precursors...respectively. Short hairpin RNA (shRNA) is conceptually similar to the microRNA precursor mentioned above and is... tissue for downstream characterization. To simultaneously address both limitations of the traditional...Artificial MicroRNAs for Multiplex Gene Silencing and Simplified Transgenic Screen. Plant Physiol 178:989–1001...

...SciFi saw the rise of literary giants such as Isaac Asimov, Arthur C. Clarke, James Blish, and John W. Campbell...strung together, it can read many base pairs simultaneously to increase specificity. This modular approach...follow a DNA-binding and DNA-cleaving strategy similar to the ZFNs, but instead of recognizing nucleotide...base editor (ABE) enzyme in 2017. ABEs rely on a similar strategy as CBE but use a ssDNA adenosine deaminase...research focuses on the use of molecular dynamics simulations and machine learning to develop a predictive ...

...your antibody can distinguish between two very similar proteins. Additionally, they require variable expression...reduced or eliminated antibody signal. If you see a similar antibody signal between your control and your knock-out...protein expression experimentally increased. The simplest version would be to use cells that do not express...you more confidence when both antibodies exhibit similar results. Unfortunately, it can be difficult to ...antibodies to your protein of interest give you very similar results, while the third looks entirely different...

... being inserted into new target sites. This is similar to how retroviruses, like HIV, replicate. Class...can be inserted between the transposon’s LTRs, similar to when packaging viral vectors.   Let’s take a...vertebrates and integrates in human cells at rates similar to retroviral vectors. The hyperactive version ...piggyBac has an integration efficiency in human cells similar to retroviral vectors. The hyperactive PB transposase...with a maximum cargo capacity of ~200 kB of DNA. Similar to piggyBac, Tol2 also prefers to integrate at ...

...and replaced it with a double-bonded oxygen. This simple enzymatic reaction enabled conversion between the...Researchers in the Xueli Zhang lab were making similar observations around the same time. (The papers ... journal on the same day.) This team created a similar construct that they called a Glycosylase Base Editor...(Huang et al., 2024). BE4max, a CBE, generated similar deletions at a lower frequency. Their data suggest...editing is still advancing rapidly. If it follows a similar path to base transition editing, we can expect ...

...adeno-associated viral vector (AAV) samples and created a simplified process, termed viral genome sequencing (VGS)...viral genome sequencing workflow. Using this simplified approach, we sequenced over 100 AAV vector preparations...routinely producing AAV. We believe that this simplified sequencing and analysis approach can be easily..., Leenay R, May AP, Paulk NK (2019) Fast-Seq: A Simple Method for Rapid and Inexpensive Validation of ...

...the goal of encouraging user contributions (using simple web forms) while also maintaining a relatively ...register with a valid email address).  One may also simply attach important references that serve to further...more powerful ways, and the advanced search page simplifies the process of building advanced multi-parametric... few years with an emission peak around 600nm?  Simply input these parameters and you’ll find matching...

...overstated, we can also learn a lot from Nature’s simpler solutions to transmitting forces over long distances...engineer it to suit our purposes? In search of simple biological machines R bodies can repeatedly ...These questions can be addressed using R bodies: simple, force-generating protein machines that can extend...

...follow multiple fluorescently tagged species simultaneously. To do so with fluorescent protein labels requires...if you know that your protein of interest has a similar spectrum. For example, here's a screenshot from...Fluorescein / Rhodamine / Cy5 (e.g. Semrock's) and the similar 4-laser combination on a confocal (405 / 488 / ...of fluorescent proteins, imaging four colors simultaneously should be pretty straightforward. Thank You...

... next cloning experiment early on. Beyond these simple but powerful display improvements, when you click...for copy and paste functionality. Sequence Similar to the plasmid maps, the linear sequences hosted...hosted here are now interactive and have similar hover over features. As with the map tab, clicking on two...Tool (BLAST) via the NCBI website to determine similarity between a given sequence and nucleotide (BLASTN...

...students focus on the application process, and simplify access to academic resources, we created STEAMid...worried about any eligibility provision, don’t guess. Simply reach out to the program coordinator with your ...through. The vast majority of programs tend to ask similar questions, thus devoting some time to writing a...Chemical Biology Program at Harvard. Roo wants to simplify access to academic resources among economically...

... thus contain complex internal cell structures similar to those of plants and animals. Unlike bacteria...mechanism of homologous recombination to facilitate simple gene replacement/mutation. Yeast expression plasmids...plasmid copy number within the bacterial cell. Similary, the specific ORI elements included within a yeast... Yeast Episomal plasmids (YEp): These are most similar to bacterial plasmids and are considered “high ...

...generating iPSCs. The generation of iPSCs is relatively simple in concept: ectopically express a cocktail of stem...factors driven by one promoter. While lentivirus has similar rates of reprogramming when compared to MMLV-derived...advantages of this method are that it’s relatively simple to implement and doesn’t require time-consuming...advantage of using RNA to reprogram cells is that it’s simple and efficient. It has a high efficiency of reprogramming...also take less time to complete. Morphology is a simple and fast to evaluate. iPSCs should form compact...

...freezer.   RNA extraction methods evolved into a simple protocol still used today There are many alternative...for RNA extraction and purification. There is one simple method that works, and variations to that method...effectively separated from DNA and proteins by a simple extraction protocol with guanidinium thiocyanate-phenol-chloroform...View our protocol for RNA extraction! Making the simple protocol even more foolproof (still without a kit...

...described above can be used for similar applications and have similar advantages, though with some caveats...the lack of an Fc region. The lack of Fc also simplifies analyses when used in structural studies and ...Alternatively, Fab fragments can be used as a block in similar scenarios. By applying unconjugated Fab fragments...

...RNA. To protect the RNA from degradation, several simple methods have been employed which modify the sugar...make your gRNA more efficient on a budget is to simply swap out several of the ribonucleotides for deoxynucleotides...resulting in less off-target events. While both have similar advantages, bridged nucleic acids with N-methyl...RNA to control the entire editing system with a simple LED light. Lighting up your gRNA with fluorescent...

...CRISPR/Cas systems, however, greatly improved the simplicity of gene activation: rather than requiring protein...CRISPR activator. While it requires a relatively simple construct, it exhibits modest levels of gene activation...compared to the SAM system. Its activation levels are similar to that of SunTag. An advantage to this method ...Shi L, Cheng L, Huang P, Yang H (2018) In vivo simultaneous transcriptional activation of multiple genes...

...indirect ELISA is very similar to a direct ELISA, aside from the detection step. Similarly, the protein antigen...Boguszewska et al., 2019.   Competitive ELISA Similar to a sandwich ELISA, the competitive ELISA is also... visualization. The indirect ELISA is used for similar experiments as the direct ELISA, the only difference...

...organization scheme for multi-part DNA assembly. Simply put, it assembles individual DNA components into...begin with a promoter and end with a terminator. A simple TU could be comprised of just three parts: promoter...assembled into TUs is key to understanding MoClo. By a similar principle, TUs can be further complexed into multi-TU...Genetic circuits Most things in biology are not so simple, and engineering complex cellular behaviors requires...

...leading the process despite the recent advance. Similarly, grad students throughout universities that are...finding money in already tight lab budgets and even simply being able to categorize all postdocs at institutions...of institutional responses to the FLSA ruling. Similarly, from purely a salary perspective, while this ... advocate for the opportunities they present. Similarly, ongoing issues that affect all scientists; such...

...elements to turn off expression of one gene, while simultaneously turning on the expression of another! What...point mutation or functional mutation in vivo: Similar to a conditional rescue experiment with a wildtype...sense (on) orientation in the presence of Cre. A similar strategy can be employed for "Cre-off" (i.e. your...Boyden Bernardo Sabatini Wenbiao Chen Liquin Luo Massimo Scanziani Scott Sternson Roger Tsien Naoshige Uchida...

...accuracy. Golden Gate assembly involves the simultaneous activities of a Type IIS restriction enzyme ...destination vector receives the assembly after similar Type IIS restriction enzyme cutting results in ...performed additional efficiency measurements using similarly sized Lambda amplicon inserts to control for any...assembly. The fidelity data can be applied to derive similar high-fidelity overhangs for any Golden Gate assembly...

...to show that they work well in plants and give similar stability to transcripts as the ones that people...we are collecting sequences that will provide a similar resource for those involved in monocot engineering...purification; the whole cloning process is so simplistic once you have your parts. That was the motivation...people to, for example, knock out a gene with simultaneous complementation, with a knockout and expression...

...getting-started guide for BLAST, which includes a simple explanation of the different BLAST programs, databases...results. The default setting is “Optimize for Highly similar sequences (megablast)”, which is very fast and ...often unreliable, we suggest using the “Somewhat similar sequences (blastn)” program for blastn so that ...options and for our suggested blastx customization. Similar to nucleotide sequences, proteins often have repeated...

...the plant genome using CRISPR/Cas9 Using pSiM24 for simplifying plant genetic engineering Using arabidopsis...seed growth on antibiotic-containing media) A similar process occurs in nature; Agrobacterium tDNA insertion... sweet potatoes (Kyndt et al., 2015)! It sounds simple, and it pretty much is, but there are some tips...

...V-K CRISPR-transposon systems function in a very similar manner to Type I-F, but instead employ a Cas12k...in single-plasmid INTEGRATE (pSPIN), which offer simple transformations for smaller cargos, and the ability... with the V. cholera INTEGRATE system follows a simple workflow: Clone spacer(s) into the CRISPR array...sequencing While CRISPR-transposons are powerful and simple to use, users should keep aware of a few important...

...hemagglutinin (HA), myelocytomatosis viral oncogene (myc), simian virus 5-derived epitope (V5), the synthetic peptide...ability to distinguish weakly expressed and/or similar proteins Through their multiple, highly specific...standard tags and can be used to distinguish highly similar proteins. For example, N-cadherin (cadherin-2) ...belongs to a huge family of proteins that are highly similar in terms of sequence and thus very difficult to...