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We narrowed to 634 results for: des.2

Showing: 181 - 200 of 634 results

  1. Hot Plasmids: Spring 2025

    Type
    Blog Post
    ...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 ...Glypicans are proteoglycans composed of a protein core, 2–4 glycosaminoglycans (GAG), and are (typically) tethered...either the optogenetic activator channelrhodopsin-2 or fluorescent reporter dTomato under the control ...Glutamate-Sensing Fluorescent Reporter (iGluSnFR) designed and improved by the Loren Looger and Kaspar Podgorski...Aggarwal et al., 2025).In this latest work, the team designed and screened a large library of iGluSnFR3 mutants...

  2. Deep Dive: Statistical Tests (Comparisons)

    Type
    Blog Post
    ...typically, normal (bell curve) or skewed (see Fig. 2). Assumptions – characteristics of a data set; ...levels of variance among data.     Fig. 2: Examples of data distribution profiles.   Parametric... positive is much higher than if you only look at 2 or 3 pairwise comparisons. Similar to family-wise ...statistical facets of reproducibility. Adv Physiol Educ. 40(2): 248-252 (2016). 0.1152/advan.00042.2016 Finch, W...Simulation Study. Front. Appl. Math. Stat. Volume 2 (2016). doi.org/10.3389/fams.2016.00002  ... been described in this blog. If your data does not meet the assumptions for the tests described above...experimental groups, but how do you determine if they deserve that oh-so coveted asterisk of significance? You...

  3. AAVs in Retinal Gene Therapy

    Type
    Blog Post
    ... whole field to a grinding stop and only now, 2 decades later, is it finally back in the limelight. On...therapy had fallen - plummeted really -  from grace 2 years prior with the death of Jesse Gelsinger in an...the other hand, have broad tropism in the eye. AAV-2, the first identified serotype and the one used in...reading 1. FDA approves gene therapy for blindness 2. Gene Therapy emerges from disgrace to be the next...(such as viral vectors) without triggering a destructive inflammatory immune response. At the time, adenoviral...follow in the footsteps of Luxturna™. However, despite promising results in preclinical studies some potential...technology: engineering of new capsid variants, designing expression cassettes to regulate and optimize ...

  4. Light Sheet Fluorescence Microscopy

    Type
    Blog Post
    ...Carballosa, et al, (2015) eNeuro, March/April 2015, 2(2) e0001-15.2015. Pubmed. Resources: http://openspim.org...orthogonal-plane fluorescence optical sectioning (OPFOS) (2). Arne Voie, David Burns and Francis Spelman focused...in LSFM. In LSFM the laser light sheet, typically 2-6  microns, illuminates only one thin plane of the...After this initial application, Stelzer’s group described the single-plane or selective-plane illumination...and acquire images of a sample from two opposing sides and merge the images, such as in the Ultramicroscope...

  5. DNA Purification Without a Kit

    Type
    Blog Post
    ...alkaline lysis plasmid miniprep Starting material: 2 mL bacterial culture Product: plasmid DNA This kit-free...protocol: 1) it solubilizes both DNA and agarose, and 2) it helps DNA stick to the glass. After binding DNA... had a similar level of sensitivity. Figure 2: Steps to purify DNA using a cellulose dipstick. Image...Academy of Sciences of the United States of America, 76 2, 615-9 . https://doi.org/10.1073/pnas.76.2.615  Zhou...plasmid purification and DNA gel extraction method described in Kim and Morrison is nearly identical to that... Image: Kim and Morrison, 2009. This method provides DNA yield and quality similar to that obtained...

  6. Viral Production at Addgene

    Type
    Blog Post
    ...digital PCR. Human Gene Therapy Methods, 25(2), Article 2. https://doi.org/10.1089/hgtb.2013.131 Wang,...(see maps section for images).     Figure 2: AAV Pro cells were transduced with either pAAV-Ef1a-mCherry-IRES-Cre...Digital PCR Method. Human Gene Therapy Methods, 29(2), 96–103. https://doi.org/10.1089/hgtb.2017.198   ...protocols, titering methods, and quality control are described below. AAV Production AAV distributed by Addgene...) are generated from lentiviral preparations (described above) that are subject to a concentration step...

  7. Opto-Nanobodies: Using Light to Manipulate Cell Signaling and Protein Purification

    Type
    Blog Post
    ...non-specific proteins, and then elute YFP.  Option #2: Opto-Nanobodies (OptoNBs) purification You skip adding...blue lights. Which option do you choose?  Option #2 may sound like fiction, but light-controlled protein...experiments.  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced...purification is now a reality thanks to OptoNBs. Recently designed by the Toettcher Lab, OptoNBs are photoswitchable... put AsLOV2 because here it was less likely to destroy the nanobody’s function than if inserted into the...blue-light induced binding. The lab used this general design scheme to generate two more OptoNBs, one against...exciting because using light to control signaling provides greater spatiotemporal resolution than pharmacological...

  8. CRISPR 101: Epigenetics and Editing the Epigenome

    Type
    Blog Post
    ...removal of H3K4me1/2 and H3K9me2 histone modifications. Like the TALE-LSD1 system described above, dCas9-LSD1...form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters and...oncogenic silencing in vivo by programmable and targeted de novo DNA methylation in breast cancer. Oncogene 34...approach for targeted DNA demethylation. Cell Discovery 2: . https://doi.org/10.1038/celldisc.2016.9 Find plasmids...which uses light to regulate transcription, also includes light-regulated histone methyltransferases and...certainly not the only CRISPR-based technology designed to alter gene expression. Fusing dCas9 to a transcriptional...epigenetic modifiers? As with any experiment, your desired outcome will determine the tool that you should...

  9. Plasmids 101: Repressible Promoters

    Type
    Blog Post
    ...immediate early gene expression. Genes Dev. 1988 Jun;2(6):718-29. doi: 10.1101/gad.2.6.718. PMID: 2843425...Targeted Gene Expression in Drosophila.” Genetics 186(2) (2010): 735-55. PubMed PMID: 20697123. PubMed Central...2022). https://doi.org/10.1038/s41598-022-07852-7 De Boer, Herman A., Lisa K. Comstock & Mark Vasser. ... in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197-214. PubMed PMID: 22129153. Additional...to the promoter and turning transcription OFF. Despite their bacterial origins, Tet systems function well...promoters can be used in the repressible manner described above, as well as the inducible manner detailed...30 years since the original Tet-Off system was described by Gossen and Bujard, both the promoter and transactivator...

  10. Recombinase-based State Machines Enable Order-dependent Logic in vivo

    Type
    Blog Post
    ...PubMed PMID: 27463678. 2. Green, Alexander A., et al. "Toehold switches: de-novo-designed regulators of gene...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...also be used to design primers to read states by qPCR or Sanger sequencing as described in the Material...hammerhead ribozyme parts are described in Nielsen et al (3) and Lou et al (4). Despite choosing proper register...machine design space is highly degenerate and often times there are multiple register designs that may...recognition sites from the register. If the RSM is designed in such a way that each possible order of inputs...

  11. Antibodies 101: ChIP

    Type
    Blog Post
    ...to (1) run a DNA gel to confirm fragment size and (2)  incubate with RNAse and Proteinase K to purify the...proteins of interest to any DNA they were bound to (2) fragmented the DNA (3) used antibodies to specifically...adjustments, to get everything just right!  Fig. 2: Goldilocks is actually celebrating a successful ChIP...immunoprecipitation (ChIP) is an extremely useful technique that provides insight into protein:DNA interactions. ChIP works...up the DNA, or enzymatic digestion. Sonication provides a truly random digestion, but is harsher and requires...both tissue or cell type, amount of protein, and desired fragment length for your output assay. A longer...

  12. Antibodies 101: Chimeric Antibodies

    Type
    Blog Post
    ...use 2 different secondaries for your antibody pair and visualize both targets in parallel (Fig. 2). For...successfully visualize both proteins.     Figure 2: Two primary antibodies with the same isotype can ...In the case above, you can’t costain with your desired antibodies because the pair belong to the same ...

  13. Plasmids 101: TOPO Cloning

    Type
    Blog Post
    ... the two DNA strands and releasing topoisomerase (2).  These days commercially available TOPO kits provide...Taq polymerase using your favorite PCR protocol.  2. Set Up TOPO Cloning Reaction: Mix together the PCR...PubMed PMID: 1658796. PubMed Central PMCID: PMC52876. 2. Novel approach to molecular cloning and polynucleotide...needed for TOPO cloning: 1. Create Your PCR Product: Design standard primers (no need to add unique restriction...

  14. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    ... analogs such as 2-fluoroadenine (2-FA), organisms with functional APT will convert 2-FA to cytotoxic ...cytotoxic nucleotides, resulting in death. In the presence of 2-FA, potent gene silencing of APT is required for...selection of plants undergoing RNAi, and LIIbeta F 1-2 RNAi, which allows assembly of intron-spliced hpRNAs...eukaryotic process where approximately 20-30 nucleotides of double-stranded RNA (dsRNA) results in downregulation... of an RNAi-inducing transgene. Typically, the desired target of RNAi is expressed as inverted repeats... and quantify actively silencing plants. Non-destructive methods, most popularly with a fluorescent reporter...Fluorescent reporter-based RNAi constructs have been designed and widely implemented in plants, such as in the...

  15. Addgene Depositors Get More Citations

    Type
    Blog Post
    ... 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010 167  Nature 3.3%  10.5%  3.2  534.1 3...Microbiology  1.6% 1.2%  0.75   125.3 In figure 2, you can see the full impact from using these weighted...plasmids deposited at Addgene still accrue on average 2 to 3 times as many citations as other papers in the... Science. For each publication, Web of Science provides basic bibliographic information and uses its vast...citations due to the journal of publication. Table 1 provides an example calculation of the weights. Table 1...

  16. The Importance of High Titer for AAV Transductions

    Type
    Blog Post
    ...drastically with lower titers. As you can see in figure 2, the expression from the 5x1011 titer was essentially... roughly 6-fold increase in expression [3, Figure 2]. Note how expression increases exponentially with...international 61.1 (2002): S9-S15. PubMed PMID: 11841606. 2. Schultz BR, Chamberlain JS. Recombinant Adeno-associated...if you end up concentrating your AAV above your desired titer and want to dilute it, err on the side of...less buffer than you would to achieve the exact desired concentration to avoid over-diluting, and make ...

  17. Addgene’s Newest Way to Speed Your Research: The Cloning Grade DNA Service

    Type
    Blog Post
    ... be directly used in the research on the SARS-CoV-2. Nine of the overall plasmid selection will be open... cloning grade DNA beta project We will ship you  2 µg of DNA in 20 µl of Tris buffer suitable for molecular...Plasmids 101 blog series Learn about the different grades of DNA  Read about the various ways to quantify...

  18. The Developmental Studies Hybridoma Bank: Over 25 Years of Antibody Sharing

    Type
    Blog Post
    ...human transcription factors such as PAX7, PAX6, AP-2 alpha and ISLET-1. In addition, we distribute monoclonal... example through the use of less toxic adjuvants; 2) generating complex antibody chips that allow assessment... which results in neutralization or targeted destruction.  Antibodies are composed of two light chains...

  19. Tips and Tricks for Using Golden Gate Modular Cloning (MoClo)

    Type
    Blog Post
    ...promoter and terminator flanking the CDS, and a Level 2 multigene vector with two other independently regulated...the sequence you are trying to clone. Figure 2: MoClo assemblies depend on compatibility of four-...sequentially for your Level 0, Level 1, and Level 2 assemblies using the plasmid sequence files provided...after each transformation. This could be as few as 1–2 colonies if you’re an experienced MoClo user, but ...manner. These videos can be especially useful when designing new primers to clone your sequence of interest... your cloning. Most sequence analysis software includes Golden Gate cloning tools that simulate the restriction–ligation...according to their concentration, size, and the desired insert-to-vector ratio using the equation in Figure...

  20. Troubleshooting Your Plasmid Cloning Experiment

    Type
    Blog Post
    ...correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers that overlap at least...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, ...coveted plasmid with no substantial delays. Design, design, design Before diving into the experimental work...experimental work. Fragments generation After you’ve designed your cloning scheme, you’re now ready to generate...from a large genome, you can recheck your primer design or optimize your primers or PCR conditions. Purifying...second and more detailed look at your experimental design. Full lawn of cells. The antibiotic in your plate...
Showing: 181 - 200 of 634 results