...    Figure 1: A 1 mg/mL control is diluted in a series ranging from 1:2 to 1:32. Each individual...trendline. R2 ranges from 0 to 1 with 1 being a perfect fit and 0 indicating that there is no linear relationship...example, Figure 1 depicts a 2-fold dilution series. Each step of the series dilutes by 1:2 for a series...series that ranges from 1:2–1:32. The specific dilution series used will depend on the expected concentration...individual step of the series is consistent at 1:2. When preparing a dilution series, use a new pipette tip for... be run in duplicate or triplicate. The closeness of the data points generated by replicate values provides... the standard dilution series in duplicate or triplicate. Replicates allows you to calculate the curve...

...Table 1 below outlines a few of the more common genetic changes found in E. coli strains. Table 1: Common...pooled library amplification. F' mcrA Δ(mcrBC-hsdRMS-mrr) recA1 endA1 gyrA96 gal-thi-1 supE44 λ-relA1 ...amber (UAG) stop codon by tyrosine insertion λ-thi-1 or thi1 Mutation in thiamine metabolism Requires exogenous... all based on E. coli K-12 and are considered BSL-1. Table 2: Lab strains of E. coli Strain Natural...plasmids, blue/white screening. F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF...) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ- JM109   General cloning and...strain for cloning repetitive DNA. endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ Δ(lac-proAB) e14- [F' traD36...

...of 0.3, as recommended by the original publication. Pro-Tip: 1. Use multiple gRNAs to target different..., as indicated by the presence of a 428 bp digestion product (lane 2). The undigested (-, lane 1) DNA ...The entire process from start to finish took about 1 month. Cas9 delivery Overview: Before any editing...lentiviral vector, lentiCas9-Blast (52962-LV, Figure 1). Some cells were then used to generate a monoclonal...rest of the cells were frozen down. Figure 1: Cas9 expression in A549 cells transduced with lentiCas9...Western blotting is a loading control. Pro-Tips: 1. It’s all about the MOI. In retrospect I used a way... pool had wildly variable Cas9 expression (Figure 1). This is a problem because Cas9 expression affects...

...promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1 Oh, J. Y., Han, J. H., ...AAV Serotypes and Their Applications in Gene Therapy: An Overview. Cells, 12(5), 1–41. https://doi.org/10.3390...constitutive promoters located in adeno-associated viral vectors. PLoS ONE, 9(8), 1–10. https://doi.org/...the selection process. Factors to think about are (1) your experimental conditions and measurements; (2...serotype, promoter, and titer.     Figure 1: Flow chart depicting a) steps for selecting serotypes...injection site than AAV2 (Watakabe et al., 2015) (Figure 1). You’ll need to consider if such properties could...

...chromosome, start, and stop. For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning of .... Table 1: An emerging family of "GPS for the genome" techniques Technique What It Locates ChIP-seq...clinical applications of CRISPR in the next section. In order to denote the NANOG enhancers from step 1 with...genetic elements like protein, RNA, and DNA (Table 1). As with GPS in the real world, coordinates alone...enhancers with the help of genome math. Figure 1: Genome math illustrated. Intersect (left) and set...histones with “GPS for proteins,” ChIP-seq from Table 1. Because of DNA’s 3D geometry and the chemical properties...experiment to identify the critical NANOG binding sites: 1. Find Enhancers that have NANOG binding sites 2. Design...

...the antibody’s dynamic range. Specifically, the 1:2, 1:1, and No dilution samples all have approximately... doing the purification and cross-adsorption in lab, consider a dilution range from 1:100–1:1,000. If ...recommended dilution and/or range (typically between 1:500 and 1:10,000 for primary antibodies). You’ll want to...from ascites fluid, try a dilution range from 1:1,000–1:100,000. Secondary antibodies For secondary antibodies...recommended dilution range is usually between 1:5,000 and 1:200,000. Check the manufacturer’s recommendations...antibodies that are not.   Table 1: Troubleshooting a western blot *indicates a diagnostic-only step Issue...reagent (usually an antibody).      Figure 1: A protein gradient. The density of the bands, which...

...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required...that occur during DNA replication. There are two ways to avoid activating MMR: 1) use a strain of bacteria...Table 1: Overview of ds- and ss-DNA Uses in Recombineering Substrate Best Suited Application Recombination... interested in science communication and in vitro diagnostics.     References 1. Costantino N, Court DL...from the 5’ end and generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ ...recombineering with an ssDNA oligo substrate.   Figure 1: Components of the Lambda Red Recombineering system...and tags.  The typical frequency of recombinants is 1 positive clone out of 104 to 105 colonies. Pro Tip...

... assembly reaction** 1 5 min., 37 ºC (2.5 μl) 687 100% 274,200 2,742,000 1 60 min., 37 ºC (2.5 μl)...References 1. Vladimir, Potapov, et al. "Optimization of Golden Gate assembly through application of ligation...expected complete sequence for the lacI/lacZ genes (1), while sequencing of white colonies showed a mixture...representative transformation plates obtained from 1-, 12- and 24-fragment assemblies of the lacI/lacZ ...lacZ cassette, and illustrates how the volume of the 1 ml outgrowth spread on each transformation plate can...fidelity studies conducted by Potapov et al. at NEB (1, 2). While T4 DNA Ligase, the mainstay of most biotechnological...over 90% for 24-fragment assemblies (Figure 3, Table 1). These improvements (5- to 12-fold increases in the...

...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems...archaea, though Class 1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible...optimal plasmids to harness the power of Class 1 systems. Class 1 CRISPR systems can be further broken down...and 17 variants. Figure 1: Overview of the CRISPR classification hierarchy. The hierarchy flows...effector complex after binding to a target. Class 1 is the most abundant of the two, comprising about ...incredible abundance, Class 1 CRISPR systems are less popular in the lab. This can be attributed to the multi-Cas...Type I CRISPR systems make up the majority of Class 1, and are the most common type of CRISPR overall. Type...

...pipetting or irregular membrane transfer. Figure 1: A loading control is a type of positive control necessary...because the loading control band is weaker in condition 1 than condition 2, the normalized values for the two...to using loading controls accurately. Assumption #1: The loading control is not perturbed by the experiment...without your knowledge? If you can’t trust Assumption #1, any change in the normalized intensity of your POI...interest band is significantly lighter in “condition 1” than “condition 2.” By eye, the loading control appears... the protein abundance axis, both blue “condition 1” points (POI and loading control) are smaller than...assumptions discussed above into question. Assumption #1: The loading control is not perturbed by the experiment...

...nervous system (CNS). AAVs are popular tools because: 1) their genomes are easy to manipulate, 2) they have... stained for a neuronal marker). Check out tables 1 and 2 below for a summary of the quantified transduction... AAV vectors. What can these AAV’s be used for?  1. Multi-color labeling of neurons Multi-color labeling... dendrites extend to. The keys to this method are 1) high levels of color diversity so that cells are ...AAV.PHP.eB 69 +/- 4% 55 +/- 7% 76 +/- 5% Table 1: Transduction efficiency of AAV-PHP.B versus AAV-PHP.eB...expression into a two part system (figure 4d):  Part 1: A cocktail of three AAV vectors expressing RGB proteins...neuronal transduction efficiency, as shown in tables 1 and 2, with a cell-type specific promoter or enhancer...

...depths (1 mm), important for certain applications such as in vivo tissue imaging.   Figure 1: Ace-mScarlet...Voltage indicators are voltmeters for cells! In this blog we will review how these indicators work, their...fluorescence or voltage indicators.   Viral Vectors and Voltage Indicators As previously mentioned...Enhanced genetically encoded voltage indicators advance their applications in neuroscience. Curr Opin Biomed...Cellular communication and function depend on potential energy. Cellular activities like action potentials...their general applications, and why viruses are essential to their use as biological tools. Why Use Voltage...Voltage Indicators? Electrical signals are integral to many facets of biology. For example, many heart...

...Inactivation of AAV Serotype 1 Vectors. Human Gene Therapy Methods, 28(1), Article 1. https://doi.org/10.1089...Figure 1).    Figure 1: Overview of key steps of AAV production, iodixanol gradient purification, and ...repeat sequences. Human Gene Therapy Methods, 23(1), Article 1. https://doi.org/10.1089/hgtb.2011.034 Benyamini...in the CNS. Molecular Neurodegeneration, 15(1), Article 1. https://doi.org/10.1186/s13024-020-00361-z ...lentivirus, AAV does not possess the ability to replicate itself and depends on helper viruses like adenovirus...herpes simplex virus to facilitate efficient replication. The most common production system, however, ...RNA genes; and the pRepCap plasmid containing replication factors and serotype-specific capsid genes.  ...

...2017. 1. Among scientists, historians have the most Twitter users. Biologists rank #8. Figure 1: Highly...that these results could be biased for two reasons: 1) they are based on the US Occupational Employment ...female to male = 0.629 (the closer this ratio is to 1, the closer males and females are to parity - the ... authorship in the United States (0.428). 4. The #1 (re)tweeted domain by scientists was Instagram Social...links to research that isn’t behind paywalls. 5. The #1 (re)tweeted science domain by scientists was Nature...mostly because of this article: “A systematic identification and analysis of scientists on Twitter.” This...from the United States Standard Occupational Classification System  and Wikipedia. 2. Social and computer...

...we observed one complication in the China data – there is a period of 1 month, from 1/22/20–2/23/20, where...was around 33% of that observed between Jan. 1 and Mar. 1, 2020. Figure 2: A simple approximation...paper. Green and red lines depict the sum of mean +1 and -1 std for each paper, respectively. The fitted...plasmid requests correspond to publications of popular papers. Figure 1: Plasmid requests by date. ... from 5/22/14 to 5/19/20). Each paper has between 1–32 plasmids and a total of 2–2,590 requests. We received...China. However, it is evident that if we believe that 1/22/20–2/23/20 are the more accurate dates of lockdown...subfields.  Plasmid requests correlate with paper publication The dataset includes 11,426 plasmid requests ...

...assembly. Journal of biological engineering. 2010;4(1):1. Pubmed PMID: 20205762. PubMed Central PMCID: PMC2822740...with minimal spectral overlap between them (Table 1). In each construct, 3 copies of the fluorescent protein... enabled labeling of particular structures (Table 1). Each construct was flanked with a CMV promoter (...construct was then introduced to HeLa cells (Figure 1). The resulting HeLa cells show robust labeling, with...fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave researchers the ability ... all four constructs into one 15 kb insert. Table 1: MXS Constructs for Labeling Cellular Structures ...maxima Tethering partner Subcellular localization 1 TagBFP 399nm/ 456nm histone 2B (H2B) Chromatin 2...

...RSM (See performance data from the indicated figures in our paper (1)) Input Plasmid Output Plasmid Fig... S9 of our paper (1)). We anticipate that, depending on the context and application, some inducible recombinase...recombinase-based state machines in living cells” (1), into a more straight-forward, concise explanation...an RSM is to sequence the register. In our paper (1), we allowed individual bacterial cells to form colonies...in the Materials and Methods section of our paper (1). Lastly, it is possible to build gene regulatory ... not. How might one implement RSMs? In our study (1), we implement RSMs in E. coli with two plasmids (...inputs to endogenous gene expression (2). Our paper (1) explains how to arrange attB-attP pairs on a register...

...incubation as it will result in very low titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more...increasing scale over the course of 1-2 weeks. Each round of amplification should result in a 10-100-fold ...repeats (ITR) present at both ends.   Figure 1: A) The structure of an adenovirus: the virion is ... 5 key steps: Construction of the rAdV plasmid (~1 week): The AdEasyTM system is the most popular method...Addgene’s Adenoviral Guide webpage, and references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid...transfected with the AdV plasmid construct from step #1 and allowed to stay in culture for up to 20 additional...CsCl are commercially available. Titer calculation (1 or 10 days) Physical titer (particle count, rAdV genomes...

...imaging of intracellular targets. Nature Communications, 7(1), Article 1. https://doi.org/10.1038/ncomms10778...by modular envelope design. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023...formin and capping protein. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023...molecules in vivo. Nature Biotechnology, 21(1), Article 1. https://doi.org/10.1038/nbt765 Lavis, L. (2016...Few Complicated Non-natural amino acids 1 Various Many Complicated   Applications Organic...fluorescent proteins, opening up new applications.   Figure 1: Live-cell labeling strategies. The...up linked to whatever molecule you choose (Figure 1). Just express the fusion protein and add a specific...

...Viral Vector Blog Posts from Addgene 1. Location, location, location! What do buying real estate and using...important factors related to location. Location (1): What tissue? Location (2): Where in the tissue/which...vectors without specific facilities (Biosafety Level-1). Other possible tools, lentiviruses for example, ...suspected cases of insertional mutagenesis in humans (1) and AAVs are non-enveloped viruses, so they are resistant...the right serotype? Keep asking questions!  Table 1: AAV Serotypes and Tropism Experiment  Detection...the vicinity of the injection. Examining serotypes 1, 8 and 9, not only do they undergo retrograde and ...types of research may be enabled by the use of AAVs? 1) Combined functional and anatomical mapping of circuits...