We narrowed to 702 results for: abo.1

...are based on the Epstein-Barr Nuclear Antigen-1 (oriP-EBNA-1) system where each vector contains a viral ...replication, OriP and the EBNA-1 sequence that encodes for the DNA binding protein EBNA-1 [7]. These two sequences...extra plasmid, pCXWB-EBNA-1 is used in the reprogramming cocktail. pCXWB-EBNA-1 lacks oriP and is unable...expressing additional EBNA-1 transiently. The transiently expressed EBNA-1 increases protein expression...pCXLE vectors listed below in Table 1 are available at Addgene. Table 1: List of pCXLE episomal plasmids,...obtained and the subsequent reprogramming success [1]. Fibroblasts need to be cultured and expanded in ...reprogramming factor cocktail, and viral protein EBNA-1. Methods for reprogramming hiPSCs from PBMCs While...

... Figure 1: Crowdfight is a community of scientists who believe in the power of collaboration and join...When a researcher needs a collaborator for a specific task, Crowdfight will: 1) Find an expert with the...our blog post about remote collaborations Read our Transferable Skills Guide article about external collaborations...needed. In the example above, for example, we fostered a formal scientific collaboration between two research...project. We think that these collaborations add enormous value. A discussion about a protocol can save weeks..., the researchers did not have access to a P3 laboratory (BSL-3), a high-security facility needed to perform...approach was useful in humans. Then, one of them heard about our online platform, called Crowdfight, and filed...

...Weight Markers. Scientific Reports,7(1). doi:10.1038/s41598-017-02693-1. PubMed PMID: 28550309. PubMed Central...plasmids that can be used to make inexpensive 100 bp or 1 kb DNA molecular weight ladders were recently deposited... PstI or EcoRV, these plasmids generate 100 bp or 1 kb DNA ladders, respectively. Unlike many commercially... gels. Get Tips on Verifying Your Plasmid Table 1: Comparing Typical Comericial Ladders with the PSU...$10 to make 1000 lanes worth of the 100 bp or the 1 kb ladder. Using a commercial plasmid prep kit increases...Learn How to Analyze a Restriction Digest References 1. Henrici, R. C., Pecen, T. J., Johnston, J. L., & ... Box Find 5 Smart Phone Apps for Lab Life Learn About the Evolution of Lab Techniques Resources on Addgene.org...

...Table 1 below outlines a few of the more common genetic changes found in E. coli strains. Table 1: Common...by tyrosine insertion λ-thi-1 or thi1 Mutation in thiamine metabolism Requires exogenous thiamine for... 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 an E. coli K-12 strain. F- λ- ilvG- rfb-50 rph-1 NEB Stable   For cloning into and storage of lentiviral...

.... The entire process from start to finish took about 1 month. Cas9 delivery Overview: Before any editing...blotting is a loading control. Pro-Tips: 1. It’s all about the MOI. In retrospect I used a way too high...to knockout BRAF in a human cancer cell line in about 1 month, which I’d say is pretty amazing. Despite...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... pool had wildly variable Cas9 expression (Figure 1). This is a problem because Cas9 expression affects...cells were transduced (MOI = 37) and selected with 1 µg/mL blasticidin for 9 days. Single cells from a ...

...    Figure 1: A 1 mg/mL control is diluted in a series ranging from 1:2 to 1:32. Each individual...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... points fit the trendline. R2 ranges from 0 to 1 with 1 being a perfect fit and 0 indicating that there...individual step of the series is consistent at 1:2. When preparing a dilution series, use a new pipette tip for...by replicate values provides useful information about the accuracy of the curve, which we will discuss...many scientific applications users aim for an R2 above 0.95.    Additional considerations When running ...

... chromosome, start, and stop. For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning...genetic elements like protein, RNA, and DNA (Table 1). As with GPS in the real world, coordinates alone...genomic coordinates into meaningful experiments. Table 1: An emerging family of "GPS for the genome" techniques...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... In order to denote the NANOG enhancers from step 1 with genome math, we’ll need a bit of shorthand from...

... 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)...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... 16 ºC) x 30 (100 μl) 78 90.7% 783 9,792 Table 1. Yields and fidelities for Golden Gate Assemblies ...

...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...

...from 1:100–1:1,000. If your antibodies come from ascites fluid, try a dilution range from 1:1,000–1:100,000...the antibody’s dynamic range. Specifically, the 1:2, 1:1, and No dilution samples all have approximately...recommended dilution and/or range (typically between 1:500 and 1:10,000 for primary antibodies). You’ll want to...recommended dilution range is usually between 1:5,000 and 1:200,000. Check the manufacturer’s recommendations...reagent (usually an antibody).      Figure 1: A protein gradient. The density of the bands, which...Here, the lanes are lightly marked on the membrane (1) using the gel as a guide. The gel is then removed...membrane is reassembled and imaged. In this example, a 1:1,000 dilution gives a high signal with low background...

...lighter in “condition 1” than “condition 2.” By eye, the loading control appears about the same between the...throws both assumptions discussed above into question. Assumption #1: The loading control is not perturbed...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... the protein abundance axis, both blue “condition 1” points (POI and loading control) are smaller than...

...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required...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...dsDNA with a recombination frequency between 0.1% to 1%, and can be increased to as high as 25-50% by designing...replication. There are two ways to avoid activating MMR: 1) use a strain of bacteria that has key MMR proteins...or 2) specially design ssDNA oligos to avoid MMR: 1) E. coli with inactivated MMR: Using E. coli with ...

... Eric Campeau 21474 pLenti CMV V5-LUC Blast (w567-1) Firefly CMV Lentiviral expression of firefly luciferase... vivo imaging or creating biosensors. Read more about BRET on our blog . Nano-Lanterns: Fluorescent proteins...

...loops 1 and 6 resulted in light controlled binding, but with opposite effects: insertion into loop 1 caused...experiments.  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced...favorite protein (YFP). You have two options:  Option #1: Affinity tag purification You tag YFP and use an ...and one against GFP. Insertion of AsLOV2 into loop 1 always generated OptoNBs with dark-induced binding... induced dark-induced blue-light induced Table 1: Summary of the effect of AsLOV2 insertion site on...Addgene blog Learn about using nanobodies to control protein activity Read about RANbodies, reporter ...reporter nanobody fusions Learn more about using the secondary nanobody toolbox for immunodetection Resources...

...promoters. Gene Therapy, 28(1–2), 56–74. https://doi.org/10.1038/s41434-020-0169-1 Oh, J. Y., Han, J. H., ...starting the selection process. Factors to think about are (1) your experimental conditions and measurements...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...AAV.  Figure 2: Comparison of AAV serotypes 1, 2, 5, 6, 8, and 9 in two different brain regions....Image modified from Aschauer et al., 2013, Figure 1. Used under Creative Commons Attribution License. ...Applications in Gene Therapy: An Overview. Cells, 12(5), 1–41. https://doi.org/10.3390/cells12050785 Murlidharan...

... regularly use the ratios below: 1 Transfer Plasmid : 1 pHelper: 1 RepCap OR 3 Transfer Plasmid : 5 pHelper...know what you think about the calculator in the comments section below. References 1. Choi, Vivian W., ...how many dishes or stacks you plan to transfect: 1 to 10 Fill in experimental details on the spreadsheet...

...in the immune system. Nature Immunology, 25(1), Article 1. https://doi.org/10.1038/s41590-023-01689-6 ... strains and lentiviral expression vectors (Table 1). The four systems Name Function Plasmid ... GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 Table 1: X-CHIME systems Proof of Concept These systems were...was lethal (LaFleur et al., 2024).   Figure 1: Schematic showing the X-CHIME approach. Figure from...genes in the immune system. Nature Communications, 10(1), 1668. https://doi.org/10.1038/s41467-019-09656-2...LaFleur is a Postdoctoral Fellow in Arlene Sharpe’s laboratory at Harvard Medical School and is interested in...K.B., Gillis, J.E., Sen, D.R., Gaudiano, E.F., Al Abosy, R., Freeman, G.J., Haining, W.N., & Sharpe, A.H...

...   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...binding to a target. Class 1 is the most abundant of the two, comprising about 90% of identified CRISPR-...types, 33 subtypes, and 17 variants. Figure 1: Overview of the CRISPR classification hierarchy. ...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...

...phosphorylated DNA termini to 3 ́-OH DNA termini (1-4). In vitro, ligases (notably T4 DNA ligase) are ... enzymology of DNA repair enzymes.     References 1. Shuman, Stewart, and Christopher D. Lima. "The polynucleotide...thermostable DNA ligase-encoding gene." Gene 109.1 (1991): 1-11. PubMed PMID: 1756968. 7. Lauer, G., et al. "Cloning... And what is ligase fidelity anyway? Let’s talk about it. DNA ligases are enzymes that seal breaks in ...to accelerate characterization of nucleic acid metabolic enzymes." Nucleic acids research 44.2 (2015): ...

...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...complication in the China data – there is a period of 1 month, from 1/22/20–2/23/20, where there were zero plasmid... 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...correspond to publications of popular papers. Figure 1: Plasmid requests by date. Parentheses depict the ...China. However, it is evident that if we believe that 1/22/20–2/23/20 are the more accurate dates of lockdown...in his laboratory. Learn more here. Additional resources on the Addgene blog Learn more about plasmid...