...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 ...

...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...

...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...

...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...

..., 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...

...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...

... 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...