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  1. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

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    Blog Post
    ...our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer team...columns are shown in red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down...Filling out and sending the deposit spreadsheet Figure 2: Example of the deposit spreadsheet showing the first...contact Addgene. We may require that you send your samples to Addgene pre-transformed in this strain. ...with instructions on how to prepare and ship the samples to Addgene and contact your technology transfer...
  2. Educational Planarian Tools for Teaching Developmental Biology Techniques

    Type
    Blog Post
    ...neoblast cells and the gene Prohormone convertase 2 (pc2) labels the central nervous system. Evaluating... characterized in an unrelated worm (C. elegans) [2]. Delivering dsRNA matching a specific mRNA sequence...223). Link: http://abt.ucpress.edu/content/79/3/208 2. Fire, S. Xu, M.K. Montgomery, S.A. Kostas, S.E. Driver...Students can produce DNA templates via PCR off of the same plasmid and use these to create riboprobes for in...WISH protocol involves preparation of the tissue sample, several washes, and temperature changes to visualize...
  3. Hot Plasmids: Spring 2025

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    Blog Post
    ...proteoglycans composed of a protein core, 2–4 glycosaminoglycans (GAG), and are (typically) tethered to ...cell-attached recordings in primary visual cortex (V1) layer 2/3 (L2/3). B) Fluorescence from axonal boutons of neurons... generation and stable cell line creation (Figure 2). Looking for more? Explore our empty and EGFP retroviral... Lentiviral backbone plasmids here! Figure 2: Addgene cloned EGFP into pAG Lenti CMV N-HA Puro ...either the optogenetic activator channelrhodopsin-2 or fluorescent reporter dTomato under the control ...
  4. Plasmids 101: Plasmid Incompatibility

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    Blog Post
    ...to plasmid loss (Diaz et al., 2015).  Figure 2: The partitioning problem. For low copy plasmids...of plasmid into a separate daughter cells (Figure 2) (Ebersbach et al., 2005). However, more recent studies...Plasmids. Microbiology and Molecular Biology Reviews 62(2): 434–464. Pubmed PMID: 9618448 Diaz R, Rech J, Bouet... Plasmids with the same replicon are incompatible because they compete for the same replication control...incompatibility due to having the same replication machinery. High copy plasmids with the same centromere-like binding...if they have the same centromere-like region, and the NTPases end up “walking” the same plasmids to one... when multiple plasmids within one cell have the same replicon and/or partitioning system. Let’s start...
  5. Selecting Your Plasmid Purification Kit

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    Blog Post
    ... environmental sustainability in mind (Table 2). Table 2: Sustainability features highlighted in plasmid...debris”; and then clean-up the plasmid DNA (Figure 2). For most kits, the steps for lysing cells and removing...together — no mixing and matching!   Figure 2: Plasmid isolation workflows are similar across kits... on plasmid preps, not all plasmid preps are the same. Your downstream application will dictate how much...plasmid DNA itself, which can happen if you leave the sample in a lysis buffer for too long or if you get a ...you mix specially coated magnetic beads into your sample and use magnets to isolate the beads for the wash...situation as you can simply add more beads to your sample. But, you’ll need to ensure you have an appropriate...
  6. CRISPR 101: Anti-CRISPR Proteins Switch Off CRISPR-Cas Systems

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    Blog Post
    ...base pair to another base pair at a specific site. 2. Temporal, spatial, or conditional control of CRISPR...difficult-to-engineer Sulfolobus islandicus rod-shaped virus 2 (Mayo-Muñoz et al., 2018). Only viral particles that...Editing of Sulfolobus islandicus Rod-Shaped Virus 2. Viruses 10:695 . https://doi.org/10.3390/v10120695...reducing editing from ~30% in control samples to 0-10% in Acr samples. Acr proteins can also be used to prevent...
  7. Photosensitizer Induced Cell Ablation with FAP-TAP MG-2I-dL5**

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    Blog Post
    ...fluorogen activating protein - dL5** in this case), and 2) a fluorogen (the TAP - targeted and activated photosensitizer...cells present 96 hours post treatment (see figure 2). Induced cell death was confirmed by TUNEL staining...PMID: 26808669. PubMed Central PMCID: PMC4916159. 2. Jacobson, K., Rajfur, Z., Vitriol, E., & Hahn, K....exposure times (5 minutes vs 10 seconds) to achieve the same level of killing as Mg-2I-dL5**. Mg-2I-dL5** also...
  8. Fluorescent Tagging of Endogenous Genes with SapTrap

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    Blog Post
    ...released (Fig. 1, Step 2-3). Since donor plasmids of the same type will produce the same unique SapI 5’ overhang...digestion of the destination vector with SapI yields 2 sites- the first site accepts the sgRNA target sequence... a gRNA vector that targets the gene of interest, 2) a plasmid to specify the reading frame of the insertion...
  9. Technique: Probe Phage Genomes for Host Binding Proteins

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    Blog Post
    ... is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...per plate) across 20 fresh selective plates. Step 2: Express random phage DNA fragments in E. coli Now...microbiology 23.4 (2015): 185-191. PubMed PMID: 25708933. 2. Cooper, Callum J., Mohammadali Khan Mirzaei, and ... biology 34 (2015): 69-77. PubMed PMID: 26275959. 2. Weigele, Peter, and Elisabeth A. Raleigh. "Biosynthesis...at the same location that their host colonies grew. If your cells of interest stick to the same location...
  10. Fluorescent CRISPR Reporters: SRIRACCHA and GEmCherry2

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    Blog Post
    ...lab found that H2B-GFP expressing cells exhibited a 2-4 fold increase in the number of endogenous indel ...Denham lab made several other small changes creating 2 other versions but these did not surpass GEmCherry2...fluorescence and in-frame fluorescence.  Figure 2: The GEmCherry reporter. Image from Højland Knudsen...resistance gene followed by the target site (the same sequence you’d like to mutate in the genome) and...
  11. Troubleshooting Your Plasmid Cloning Experiment

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    Blog Post
    ... working with is correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...colonies (5-10) and grow them in a small culture volume (2 or 3 ml) containing the corresponding antibiotic, ...Remember to quantify the DNA concentration of all the samples by gel or by nanodrop to assemble the  fragments...
  12. ReFigure: Save Scientific Figures into Dashboards and Share Your Insights

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    Blog Post
    ...literature? Very often the data of interest are in 1 or 2 figures from one paper and then another figure in ...ReFigure by creating ReFigures and answering this 2 minute survey! You can also find ReFigure on Twitter...another paper. A large fraction of readers cover the same ground again and again as they don’t save the insights...disease or therapeutic Side-by-side comparison of the same experiment from different cell lines/model systems...
  13. Tips for CRISPR Gene Editing in Mice

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    Blog Post
    ...groups (4; brighter than control, 3; same as control (Cetn1/sgRNA1), 2; darker than control, 1; very dark...you can hold off on genotyping until the pups are 2-4 weeks old and use the ear tag method (creating different...PMID: 24253446. PubMed Central PMCID: PMC3875854. 2. Mashiko, Daisuke, et al. "Generation of mutant mice...future.  Many thanks to our guest blogger Samantha Young. Samantha Young is a Medical Writer with a PhD in... This post was contributed by guest blogger Samantha Young. The use of CRISPR/Cas9 for gene editing has...technique, you can assess several sgRNA’s at the same time, and choose the most effective ones to increase...
  14. New and Upcoming Viral Vectors - June 2019

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    Blog Post
    ...  AAV9, AAVrg pAAV-hSyn-DIO-mCherry 37825 AAV1, 2, 5, 8, 9 (20 µL size serotype testing) pAAV-CAG-GFP...size serotype testing) pAAV-CAG-GFP 50465 AAV1, 2, 5, rg (20 µL size serotype testing) pAAV-hSyn-EGFP...size 20 µL samples of control AAV that scientists can use for serotype testing. These samples are available...serotype testing jGCaMP7c for calcium detection PSAM4 GlyR for chemogenetic inhibition Flp expression ...localization. The Sternson lab recently deposited PSAM AAV, which encode engineered ligand-gated ion channels...Plasmid Serotype Name 119741  AAV5  AAV SYN flex PSAM4 GlyR IRES EGFP Recombinase AAV Finally, we are... Plasmid Serotype Name 119472  AAV5  AV SYN PSAM4 GlyR IRES EGFP   Biosensors Plasmid Serotype...
  15. RNA Interference in Plant Biology: New Tools for an Old Favorite

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    ... analogs such as 2-fluoroadenine (2-FA), organisms with functional APT will convert 2-FA to cytotoxic ...selection of plants undergoing RNAi, and LIIbeta F 1-2 RNAi, which allows assembly of intron-spliced hpRNAs...nucleotides, resulting in death. In the presence of 2-FA, potent gene silencing of APT is required for survival...between gene silencing and knockout methods of the same gene. However, recent work has called this prevalent...
  16. Starter Guide to induced Pluripotent Stem Cells (iPSCs) Part 2:  Reprogramming and Transdifferentiation

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    ...revert back to a pluripotent stage (iPSC formation) [2, 3]. The iPSCs then proliferate and redifferentiate... more common delivery methods include retrovirus [2], lentivirus [4], adenovirus [5], Sendai virus [6]...2006. 441(7097): p. 1061-7. PubMed PMID: 16810240. 2. Takahashi, K., et al., Induction of pluripotent stem... Pluripotent Stem Cells. Cell Stem Cell, 2015. 17(2): p. 178-94. PubMed PMID: 26189426. 15. Si-Tayeb, ...induced pluripotent stem cells. Stem Cell Rev, 2015. 11(2): p. 242-53. PubMed PMID: 25578634. PubMed Central... to regeneration. Nat Rev Mol Cell Biol, 2011. 12(2): p. 79-89. PubMed PMID: 21252997. 37. Merrell, A....human induced pluripotent stem cells from urine samples. Nat Protoc, 2012. 7(12): p. 2080-9. PubMed PMID...
  17. Antibodies 101: Flow Cytometry Controls

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    Blog Post
    ...positive population. Figure 2: An FMO gating example. The FMO sample (A) can be used while setting ...otherwise be difficult to separate out. In Figure 2, you can see that, without the FMO, it would be difficult...It will be from the same host species, have the same Ig subclass, and be in the same fluorophore (e.g.,...controls An isotype sample is prepared very similarly to a FMO sample: you make a sample from a combined ...your samples. The single color controls should be used as a starting point, to preserve your sample, and...experiment. To make an FMO sample, you take a subset of your experimental samples, combine it into one well...controls will help you save on sample volume, so you can use most of your samples for flow analysis rather ...
  18. Antibodies 101: Reading a Flow Plot

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    Blog Post
    ...A-B+ cells. If you look at the dot plot in Figure 2, you might assume that all cell populations are positive... the different populations. In the case of Figure 2, one population is B- and another two are B+. Our ...are in the lower right corner.      Figure 2: This flow plot has two populations that are B+ and... are represented as a series of dot plots in the same order. However, in an actual flow cytometry run,...parallel.  Let’s imagine we’re looking for cells in a sample that are negative for marker A and positive for... proxy for nucleic complexity of the cell.  The sample will have been labeled using a panel of markers... dots. In a contour plot, each line contains the same number of events. Thus, contour lines that are close...
  19. The AAVantages of AAV in CRISPR Screens

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    Blog Post
    ...019-12449-2 Pupo, A., Fernández, A., Low, S. H., François, A., Suárez-Amarán, L., & Samulski, R. J. (2022...handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires a less involved setup when working...infection rates in your experiment. Figure 2: Visual representation of common AAV serotypes used...vivo. The general process of these screens is the same: Deliver CRISPR library to cells of interest. Select...
  20. Production of Virus in Insect Versus Mammalian Cells

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    Blog Post
    ...) 38 kb Cells HEK-293T Sf9 Time to collection 2–4 days 5–10 days Purification Ultracentrifugation...Centrifugation Titration time ~1 day (AAV); ~1 week (lenti) 2–10 days Initial titers Higher Lower Resources... a factory to produce adeno-associated virus type 2 vectors. Human Gene Therapy, 13(16), 1935–43. https..., so the amount of cloning required is about the same between insect and mammalian systems. Baculovirus...P1 virus, so that if you ever need to remake the same virus, you can skip the first round of production...
  21. Neuronal labeling with Spaghetti Monster

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    Blog Post
    ...experiment using smFPs and standard neuronal tracers. 2. Visualization of neuronal sub-cellular structures...similar proteins. For example, N-cadherin (cadherin-2) is a postsynaptic cell adhesion protein that plays...PMID: 25915120. PubMed Central PMCID: PMC4573404. 2. Shepherd, G.M. (2004). In "The synaptic organization...super-resolution STORM imaging, and electron microscopy. Sample preparation for these techniques sometimes makes...smFPs retain their ability to be detected after sample preparation better than standard proteins tags ...
  22. Evolution of Lab Techniques

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    Blog Post
    ... is the genetic test used for sickle cell anemia (2). Southern blotting, in conjunction with restriction...program) can then reproduce the DNA sequence (Figure 2). Accuracy and accessibility led to the widespread...biology98.3 (1975): 503-517. PubMed PMID: 1195397. 2. Chang, Judy C., and Yuet Wai Kan. "A sensitive new...searching for), then the probe will bind to the sample thereby labeling it. The label can then be visualized...strength of the signal in comparison to a control sample. Southern blotting applications range from confirming... MstII and southern blotting are conducted on a sample from an individual without sickle cell anemia, ... largely replaced by real-time PCR to answer the same experimental questions. Real-time PCR detects and...
  23. FLEx Technology and Optogenetics: Flipping the switch on gene expression with high spatial and temporal resolution

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    Blog Post
    ...upstream of an opsin gene (e.g., channelrhodopsin-2, ChR2) fused to a reporter gene (e.g., mCherry) to...the correct orientation to be transcribed (Figure 2) (Sharma and Zhu, 2014). How does FLEx enable strong... Abdallah, Khaled, et al. "Adeno-associated virus 2/9 delivery of Cre recombinase in mouse primary afferents...Deniz, et al. "A FLEX switch targets Channelrhodopsin-2 to multiple cell types for imaging and long-range ...general anesthesia (Taylor et al., 2016). These very same neurons also play a central role in motivated behaviors.... Hirsch, Matthew L., Sonya J. Wolf, and R. J. Samulski. "Delivering transgenic DNA exceeding the carrying...
  24. Antibodies 101: Introduction to Gating in Flow Cytometry

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    Blog Post
    ... intensity of the cell’s illumination (see Figure 2). H describes the maximum signal strength, while W...SSC-A, so most people use that setting. Figure 2: As the cell passes through the laser beam and causes...scale in proportion (while H and W do not; see Figure 2), single cell events will show up in a straight line...When using flow cytometry to analyze your samples, it is necessary to set up a sequence of gates to be...the area under the resulting curve. By default, a sample on the flow cytometer (before you set the first...Practical example: lymphocytes in a mouse tumor sample You made it! So much about the theoretical background...gated out my lymphocyte population from a tumor sample. Many cell types can be found in the tumor microenvironment...
  25. Troubleshooting and Optimizing a Western Blot

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    Blog Post
    ...dynamic range. Specifically, the 1:2, 1:1, and No dilution samples all have approximately equally dense... that all look exactly alike.      Figure 2: Reagent gradient. Here, the lanes are lightly marked...guide. The gel is then removed from the membrane (2) and the membrane is cut so each lane is its own piece...in the bands, you may want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to ...conditions are 4 °C overnight or room temperature for 1–2 hours, there is some evidence that antibodies may ...time.   Samples can run into empty neighboring lanes, so load your gel to ensure your samples and reference...protein-based buffer (FBS, milk, etc.) against the same amount of sample.  If you’re using chemical blockers, you...
  26. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

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    Blog Post
    ...Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow 3.2. ...M-CFP expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate... each fluorophore. Third, Brainbow-1 and Brainbow-2 each contained a “default” state; for example, Brainbow...processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained...three loxP variants, but the principles remain the same. The first fluorescent protein in the construct,...
  27. Fluorescent Proteins 101: Introduction to FRET

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    Blog Post
    ... fluorophore excitation by absorption of a photon 2. Energy transfer from the excited donor directly to...PMID: 24194740. PubMed Central PMCID: PMC3810607. 2. Bajar, Bryce T., et al. "A Guide to Fluorescent Protein...PubMed PMID: 10500161. PubMed Central PMCID: PMC18018. 2. Miyawaki, Atsushi, et al. "Fluorescent indicators...contain the donor and acceptor fluorophore on the same protein and are useful when a process affects the...
  28. Pathways Over Time Plasmids Engage Students in Functional Genomics Research

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    ...research experiences to undergraduate students (1, 2), but educators face significant challenges in designing... by western blotting in other experiments (Figure 2).   Complementation reveals conservation of function...Biology Education: A Call to Action, Washington, DC. 2. President's Council of Advisors on Science and Technology...fission yeast, Schizosaccharomyces pombe, have the same function as their counterparts in S. cerevisiae....
  29. Adeno Associated Virus (AAV) for Cell and Gene Therapy

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    ...AAVs in the clinic Thus far, AAV vectors AAV1, AAV-2, AAV1-AAV2 hybrids, AAV-6, AAV-7, AAV-8, AAV-9 and...neutralizing antibodies against some AAV types, AAV1, 2, 3 and 5 are thought to be as high as 70% among the... 10.24 (2003): 1999-2004. PubMed PMID: 14566358.  2. Hirsch, M. L., et al. "Self-complementary AAV mediates... PubMed Central PMCID: PMC3152950. 3. Jacobson, Samuel G., et al. "Improvement and decline in vision with...
  30. Making CRISPR Plasmids Using Fragmid

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    Blog Post
    ... of components, such as your CRISPR enzyme or Pol 2 promoter. The options available in each class, or ...overhang specific to the type of module it is (Pol 2 protomer, Cas protein, ect...). Whatever individual... (KO, CRISPRa, CRISPRi, editing)   Figure 2: Selecting module fragments on the Fragmid website...(GG) cloning approach. Individual modules and a sample ligation are depicted. (Bottom) Schematic depicting...
  31. Advice for Starting a Biotech Company from a Grad Student turned Entrepreneur

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    ...go anywhere. The team should initially consist of 2-3 individuals that are aligned in vision and outcomes... the technology. This transition takes around 1 – 2 years. The product is the measure of success. Technology... into Sample6 as graduate students in Dr. Jim Collin’s Lab in 2009. From 2009 to today, Sample6 has changed...leadership, and grown to 25 members.  What is Sample6Sample6 is an enrichment-free bacterial pathogen detection...current VP of Business Development & Operations of Sample6, for advice on how to take an idea developed during...etc. What project in the Collins lab inspired Sample6? The initial work focused on using bacteriophages... the focus of the application was changed, but Sample6 still leverages the core engineering and scientific...
  32. Three Key Considerations for Precise Neuronal Targeting Using AAV Technologies

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    ... characteristics of select AAV serotypes in Table 2 from the paper. Delivery of AAV into the brain can...routes for neuroscience see Table 1 from the paper. 2. Manipulate your cell of interest: Cell-type specific...intravenous injection. Even when injected into the same place in the brain, certain serotypes spread further...
  33. Antibodies 101: Polyclonal Antibodies

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    ...animal is given additional booster immunizations every 2-3 weeks to increase the antibody titer. The titer ...level of antibodies is reached - typically within 2-4 months. After the animal has the desired level of...recognize the same region, or epitope, of the target protein. These antibodies are also all the same isotype...heterogeneous mixture of many antibodies that recognize the same protein. In the immune system, antibodies are produced...peptide sequence derived from this protein. At the same time, an adjuvant such as KLH or Freund’s adjuvant...clones will still recognize your protein. For the same reason, they’re also useful in experiments where...experiment, you should either order several vials of the same lot of polyclonal antibody, or select a monoclonal...
  34. Recombinase-based State Machines Enable Order-dependent Logic in vivo

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    ...certain states of interest. For example, in Figure 2 below we present a two-input, five-state GRSM register...linking RSM inputs to endogenous gene expression (2). Our paper (1) explains how to arrange attB-attP...Science. 353, aad8559 (2016). PubMed PMID: 27463678. 2. Green, Alexander A., et al. "Toehold switches: de-novo-designed...states in a sample; instead, it will suggest the predominant state of registers in a sample based on a ... use the same recognition site array on the register, and all three-input GRSMs use the same recognition...implement the same gene regulation program. We recommend testing multiple register designs for the same gene regulation...If an attB-attP pair is aligned (sites facing the same direction) on a register, then the result of their...
  35. Cellosaurus: A Cell Line Information Database

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    Blog Post
    ...groups, such as their vaccine production, SARS-CoV-2 research, or adenovirus packaging cell lines dataset...-Line Knowledge Resource. J Biomol Tech. 2018; 29(2): 25-38....hybridoma, etc.), as well as sex and age at time of sampling if available. STR profiling for human, mouse, ...
  36. New Tool for Lineage Tracing: The ClonTracer Library

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    ...represent the preexisting resistant subpopulation 2. Resistant cells among the separate replicates would ...medicine 21.5 (2015): 440-448. Pubmed PMID: 25849130. 2. Hata, Aaron N., et al. "Tumor cells can follow distinct...replicates would contain the same labels - i.e. they would always come from the same parent cells that represent...replicate groups, and treated these groups with the same drug regimen, they would find one of two outcomes...population (all barcodes can be amplified using the same sets of forward and reverse primers). The abundance...
  37. Hot Plasmids: Winter 2025

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    ...resistance to the herbicide chlorsulfuron (Homo line 2) or wild type (WT), two weeks after herbicide was ... CLIPf tag and a C-terminal SNAPf tag; see Figure 2), the authors developed a cost-effective expression... when associated with microtubules. Figure 2: CLIP-SNAP fusion proteins for single-molecule FRET...recombinant antibody in human postmortem brain samples. Results show positive staining in tissues from...
  38. Plasmids 101: Protein tags

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    ...(you generally don’t want more tag than protein), 2-3 tags is increasingly common. Tandem affinity purification...TEV cleavage site (more on that in a moment), and 2 ProtA IgG-binding domains. TAP has since come to encompass...found in this paper (available at Addgene).   Table 2: Protease recognition sites commonly used with tags...protein since the enzyme cleaves the SUMO tag in the same manner as it would have in the context of a cell...
  39. Viral Vectors 101: Inverted Terminal Repeats

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    ...ITR, which can either be “flip” or “flop” (Figure 2). Additionally, the ITR contains a 4-nucleotide Rep...site for Rep proteins (Savvy et al. 2013). Figure 2: AAV2 wild type ITR in flip and flop orientation. ...cis-acting elements, which means that they are in the same DNA molecule as the gene that they package. In a.... M., Dobbins, A. L., Li, C., Hirsch, M. L., & Samulski, R. J. (2020). Adeno-Associated Virus Serotype-Specific...
  40. A New Optogenetic Tool Based on AraC Controls Gene Expression with Blue Light

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    Blog Post
    ...demonstrated through living images created by the team (Fig. 2). These images are based on the concept of bacteriographs...spatially precise the activation is.   Figure 2: A bacteriograph of Michelangelo's "The Creation of...References Romano E, Baumschlager A, Akmeriç EB, Palanisamy N, Houmani M, Schmidt G, Öztürk MA, Ernst L, ...
  41. Summer SciComm: Science Storytelling

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    ... (1) the struggle between two opposing forces or (2) the result of competing desires or (3) the presence... an introduction, and a resolution.   Fig. 2: The structure of a story - not just for fiction! ...ending, we can still get invested in the journey? The same thing applies for science storytelling. You can ...
  42. Antibodies 101: Conventional vs Spectral Flow Cytometry

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    ...fluorophore from another within a fully stained sample. Figure 2 is an example of the emission spectra for ...another during spectral unmixing.     Figure 2: The full emission spectra for BV421 and BV711 on ...stained samples. If you are using conjugated dyes, it is critical that your controls and your sample antibodies...unmixing if your sample is stained with a PE/Cy7 antibody from a batch that is not the same as your single...allows the user to use the autofluorescence in a sample as a fluorescent marker. Given that the level of... varies, it is critical to include an unstained sample for each tissue type you are running in your panel...control(s) Compensation or “reference” controls are samples stained with a single fluorophore. They’re required...
  43. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

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    ... of permeabilization and membrane integrity (Fig. 2). For example, saponin interacts with cholesterol ...at too high a concentration.      Figure 2: Examples of different permeabilization strategies and... permeabilize a sample. But you can also use organic solvents after fixing your sample with a cross-linker...the point?  Fixation allows you to freeze your samples in a given state or point in time. You want to ...processes while maintaining the structure of your samples, the relationships between cellular components,... molecules and it is great for preserving your samples and maintaining cellular and tissue morphology....formaldehyde solutions.  Along with preserving your sample, cross-linking can block the epitope of some targets...
  44. Form Your Own Peer Mentoring Group: A How-To Guide for Scientists

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    ...managing to get together. If you all work at the same place, a 1-2 hour lunch on a regular schedule might work...The groups in these programs typically consist of 1-2 mentors and 3-5 mentees who meet together for an 8...Allows all group members to lead and learn at the same time Broadens the network of the participants Can...
  45. Summer SciComm: Preprints

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    ... to share information about the emerging SARS-CoV-2 virus quickly and openly. By sharing results faster...Open Scientific Communication Ecosystem Fig. 2: The scientific ecosystem, enriched by open access...communication. Researchers often post a preprint at the same time as, or slightly before, submitting the manuscript...plasmids that were used in a preprint provides all the same benefits as sharing those plasmids alongside a journal...
  46. Antibodies 101: Choosing the Right Isotype

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    ... reduce the background staining (Table 2).      Table 2: Secondary antibody selections for a four-antibody...affinity binding, such as immunoprecipitations. Your sample species should also be considered when choosing...immunoglobulins naturally present in the tissue sample. There are species-on-species staining protocols...secondary antibodies that are all derived from the same host species and have been cross-adsorbed. The cross-adsorption...using mouse IgG2a and rat IgG1 antibodies in the same application, using a Goat anti-mouse IgG2a cross-adsorbed... species, and secondary antibodies raised in the same species that have been cross-adsorbed to reduce ...
  47. PCR Applications for Copy Number Variation Assays

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    ...mean you have one copy of the gene; an RQ value of 2 means you have two copies, and so on and so forth....quantitative PCR. Expert Rev Mol Diagn. 2005 Mar;5(2):209-19. doi: 10.1586/14737159.5.2.209. PMID: 15833050...to a reference sequence present in the same amount in all samples (usually to a ubiquitously expressed,... reference sample with two copies of the gene (Ma, 2014.) As with all qPCR assays, samples, standard curves...housekeeping’ gene), and quantified relative to a 'normal' sample with known copy number set as the expression baseline...since you’re looking at the amount of DNA in your sample relative to your normal control - you can set up...number (RQ value) matches the copy number of your sample. In that case, an RQ value of one (or, more realistically...
  48. The PAM Requirement and Expanding CRISPR Beyond SpCas9

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    ...is unique compared to the rest of the genome and (2) the sequence is located just upstream of a Protospacer...pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from species other...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi....and crRNA (or synthetic gRNA) are derived from the same species. Expanding the CRISPR toolbox The isolation...
  49. Twenty Years of Sharing: Expanding Accessibility

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    Blog Post
    ...China, Korea, Japan, Brazil, India, and Mexico. Table 2: Addgene distributors Distributor Partnership...distribution after we partnered with them (Figure 2). In others, the distributor helped solve an issue...temperature for the duration of their journey. Figure 2: Annual distribution of items to countries with a ...that available and accessible weren’t always the same thing, shaped both Addgene’s founding and our growth...
  50. Illuminating Epigenetics with A FRET Based Biosensor

    Type
    Blog Post
    ...FRET signal back again. The Ting lab constructed 2 versions of this reporter to target the K9 (pcDNA3... 525.7570 (2015): 533-537. PubMed PMID: 26352475. 2. Bygren, Lars O., et al. "Change in paternal grandmothers...corresponding gene products due to the same region, but not the same exact genes, being improperly silenced.... This is how most cells in the body contain the same DNA but have very different structure and function...present without any DNA changes, and both involve the same region of chromosome 15. They both result from the...
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