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  1. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

    Type
    Blog Post
    Published
    Aug. 9, 2016, 2:30 p.m.
    ...identify the critical NANOG binding sites: 1. Find Enhancers that have NANOG binding sites 2. Design a CRISPR...stem cells (ESCs). Your ChIP-seq finds just over 13,000 significant binding peaks for NANOG in the human ...on Addgene.org Find CRISPR Plasmids for Your Research Check out Our CRISPR Guide Find Validated gRNAs..., start, and stop. For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning of the ...emerging family of sequencing techniques function as a kind of “GPS for the genome” to compute coordinates for...DNase-seq DNA (regions that are accessible for binding) Google Maps: Enhancers and genome math Suppose...Suppose that you wish to use Google maps to find all coffee shops near your house, excluding Starbucks. ...

  2. Components of CRISPR/Cas9

    Type
    Blog Post
    Published
    Feb. 2, 2016, 5 p.m.
    ...variant thereof. This individual protein has all the components necessary to: 1. Bind to a guide RNA The ...many possible loci. Without binding to the guide RNA, Cas9 cannot cut. 2. Bind to target DNA in the presence...protospacer adjacent motif (PAM) Cas9 endonuclease binding to the target genomic locus is mediated both by...the guide RNA or a PAM sequence, Cas9 will neither bind nor cut the target. Cas9 homologs from different...domain near the center of the protein. Upon target binding, Cas9 undergoes a conformational change that positions... SpCas9 D1135E variant 3' NGG (reduced NAG binding) SpCas9 VRER variant 3' NGCG SpCas9 EQR...) or single guide RNA (sgRNA). Throughout the remainder of this post, we’ll refer to this RNA complex ...

  3. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    Published
    April 11, 2017, 2:30 p.m.
    ... the Jaffrey lab used SELEX to find a 98 nt RNA aptamer that would bind the fluorogen and increase its...GFP Resources at Addgene.org Find the Spinach Plasmids at Addgene Find Broccoli Plasmids at Addgene View...What is an aptamer? Nearly 30 years ago, two independent groups, led by Jack Szostak and Larry Gold, developed...selecting and amplifying RNA sequences that could bind very specifically to target molecules. Using a technique...non-nucleotide targets. These RNA molecules, which could bind their targets with high specificity and affinity...next two decades, these nucleotide-based ligand binders would prove to be highly adaptable tools. The...frequently undergo conformational changes after target binding, aptamers are highly customizable in terms of how...

  4. Targeted Mutagenesis with EvolvR

    Type
    Blog Post
    Published
    Feb. 21, 2019, 1:15 p.m.
    ...editing window of ~17 nt from the nick site, which was expected since the processivity window of PolI ...thereby increasing the editing window of EvolvR to ~56 bp. A larger editing window also increased the likelihood...of interest with dCas9 fused to the Activation Induced Cytidine Deaminase (AID) enzyme. AID, however, ...nCas9 nicks and then dissociates from. PolI3M then binds the nicked DNA, and extends it from the 3’ end, ...its target DNA (left). Cas9 dissociates and PolI binds (middle) and extends from the 3' end of the nick...increase the mutation rate and extend the editing window of EvolvR: (1) two additional mutations to PolI3M...generation, and (2) the addition of the thioredoxin-binding domain (TBD) from bacteriophage T7 DNA polymerase...

  5. Plasmid Tools for Microbiome Studies

    Type
    Blog Post
    Published
    June 27, 2019, 12:25 p.m.
    ...designed to bind and detect nearly any RNA sequence. Downstream of the hairpin is a ribosome binding site (...under control of the arabinose-inducible promoter. Cells are first induced with arabinose to begin expressing...Addgene Blog Find microbiology blog posts Read about synthetic biology on the blog Find all of our Plasmids...for in situ genetic tools for microbiome studies.  Find the MAGIC plasmids here Paper-based synthetic biology...blocks translation by sequestering the RBS, but binding of the “trigger RNA” (in this case, biomarker RNA...transcripts of interest is a benefit of this method. Find the toehold switch microbiota/biomarker sensors ...antibiotic resistance markers, promoters, and inducible gene expression systems in bee gut microbiome ...

  6. SARS-CoV-2/COVID-19 Detection Methods Based on CRISPR/Cas

    Type
    Blog Post
    Published
    May 5, 2020, 1:15 p.m.
    ...complex binds to the target sequences, Cas12a activity gets activated. This unleashes indiscriminate ssDNA... complementary crRNA sequence to bind to the sample and upon binding, gets activated. This activation ...that have no mismatches bind to the immobilised target sequences. Upon binding, changes in fluorescence...streptavidin which can bind to the biotinylated targets. They also tried to confirm target binding on agarose gels... Two Cas12a-crRNA complexes are designed, each binding to one strand of the reverse-transcribed target... and are added separately. Using two crRNAs that bind to two regions improves sensitivity of the assay...amplification by the RPA primers, the Cas12a-crRNA binds to the complementary region in the target sequence...

  7. Fluorescent Proteins 101: When GFP lets you down

    Type
    Blog Post
    Published
    Aug. 23, 2018, 12:05 p.m.
    ...that bind endogenous fluorescent co-factors can be used. Examples include the LOV domains that bind flavin...protein that binds bilirubin (Kumagai, 2013). Next to these naturally occurring co-factor binding proteins...molecules can be indirectly visualized by using protein domains that specifically bind the molecule of ...proteins, systems wherein a protein binds a synthetic, fluorescent analogue have been engineered (Thorn, 2017...is big GFP is a 28 kDa protein that resembles a cylinder with a length of 4.2 nm and a diameter of about...genetically encoded probes are based on cofactor binding proteins as described above. The smallest tags ...tags are peptides that are engineered to bind specifically and with high affinity to fluorophores. Examples...

  8. Antibodies 101: Introduction to Antibodies

    Type
    Blog Post
    Published
    Jan. 19, 2021, 2:15 p.m.
    ...immune system useful as a research reagent? Let’s find out! What is an antibody? Antibodies, also known...system, antibodies are produced by B cells. They bind to proteins on the surface of extracellular pathogens...is an Antibody?    The ability of antibodies to bind proteins is useful for research applications as ...Antibodies have two regions: the Fab, or antigen-binding region, and the Fc, or crystallizable region. Two...determine what antigen the antibody recognizes and binds to.   Figure 1: Labeled diagram of an antibody...variability between lots is a big concern. You can find more details about polyclonal antibodies on our ...antibody molecule is not necessary for antigen binding, the variable regions alone can be generated as...

  9. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    Published
    Oct. 12, 2023, 1:15 p.m.
    ...transgene expression-induced toxicity in NHPs (Hordeaux et al, 2020; Hudry et al, 2023; Hinderer et al, 2018).... 2023 * indicates viral preps available. Mechanisms of crossing the BBB are indicated in parentheses..., the suitability of a systemic capsid for an individual application is dependent on a number of factors...like CREATE (Deverman et al., 2016), M-CREATE (Ravindra Kumar et al., 2020), iTransduce (Hanlon et al.... vg total in mice) may increase the chances of inducing an immune response against the capsids or transgenes... CLOVER repository of published systemic AAVs to find commonly used vector cargo designs and administration...deposited by the Maguire lab. In mice, it is sex-independent and does not show significant differences in ...

  10. Three Tips to Organize Your Lab Notebooks in the New Year

    Type
    Blog Post
    Published
    Jan. 2, 2014, 3:44 p.m.
    ...handwriting or trying to find to where the binder was moved. You’ll have peace of mind knowing the information... and post-its to make finding your most used protocols & references easy to find It’s happened to all ...you can’t find the molecular cloning guide you were sure was in one of those four white binders. Thirty ...on the spine. For binders of protocols and data, consider using color coded binders and tabs (for different...Start by gathering that pile of lab notebooks and binder on your desk. Arrange them in chronological order...tasks, set deadlines, and arrange automatic email reminders. These programs can be synced to your online calendar...to other notes, and use the search bar to quickly find what you’re looking for. Tip 3: Share protocols ...

  11. Hot Plasmids and Viral Preps - March 2021

    Type
    Blog Post
    Published
    March 23, 2021, 1:15 p.m.
    ...it easier for you to find and use the plasmids you need. Here's what you'll find in this post: Epigenetic...the auxin inducible degron (AID) system in C. elegans By Alyssa Cecchetelli The auxin inducible degron (... a light-inducible CRISPR system developed by Taekjip Ha’s lab where Cas9-gRNA complex binds the target...assembly Tracking cell-cell interactions New auxin inducible degron system for C. elegans New CRISPR plasmids..., 2021).   Schematic showing the auxin-inducible degron system. Image from Ashley et al., 2021....spatiotemporal expression pattern and an auxin inducible degron (AID) sequence attached to your protein...complex. In the presence of auxin, this TIR1 complex binds to the degron sequence ultimately leading to the...

  12. How to Design Your gRNA for CRISPR Genome Editing

    Type
    Blog Post
    Published
    Sept. 24, 2020, 1:15 p.m.
    ...100nt window upstream of the transcription start site (TSS), while for CRISPRi, a ~100nt window downstream...Basics with Our CRISPR Guide Find CRISPR Plasmids for Your Research Find CRISPR Pooled Libraries for Your...updates on how best to design gRNAs. You can also find validated gRNAs for your next experiment in Addgene's...important considerations for designing your gRNAs. For indels, it's not so important what location in the gene...joining (NHEJ) often leads to the generation of indels and thus frameshifts that disrupt the protein-coding...consuming process and should not be undertaken lightly! Indeed, truly achieving the gold standard requires not...editors, the intended edit must be in a 5 - 10 nt window relative to the PAM, and bystander edits are possible...

  13. Hot Plasmids: Winter 2025

    Type
    Blog Post
    Published
    March 11, 2025, 1:15 p.m.
    ... our Hot Plasmids articles.  Here's what you'll find in this post: Optimized prime editing in dicots...single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies for neurodegeneration...potential to improve rapid and precise plant breeding. Find dicot prime editing plasmids here! Vu, T. V., Nguyen...kinesins but for many other proteins of interest too! Find CLIP, CLAP, and SNAP plasmids here! Smith, E.R....2024.12.20.629623.  A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic...and more responsive genetically-encoded voltage indicator (GEVI) than previous versions. ASAP5 can be imaged...The initiation site identified in panel (C) is indicated; red line is exponential fit. Adapted with permission...

  14. Chromoproteins: Colorful Proteins For Molecular Biology Experiments

    Type
    Blog Post
    Published
    Feb. 4, 2020, 2:15 p.m.
    ... Addgene blog Find our Fluorescent Protein 101 blog series Learn more about FRET Find protocols and tips...Addgene.org Find fluorescent proteins for your research Read our Molecular Biology Reference Find plasmids...phytoplankton, and colorful corals, to name a few. What’s behind these sights are an abundance of biological molecules...limited or for bioart and teaching applications. Find a chromoprotein for your experiment! Eukaryotic ...contain a toxin gene (regulated by a rhamnose-inducible promoter) and a chromoprotein gene. Allelic exchange...expressed as it is under the control of a rhamnose-inducible promoter. The resolution step leads to the desired... backbone. To do this, rhamnose is added which induces expression of the toxin. Any cells that grow after...

  15. Cloning Mammalian Cells with the Agarose Method

    Type
    Blog Post
    Published
    Sept. 7, 2017, 12:17 p.m.
    ...blogger Iris Lindberg, Professor at the University of Maryland School of Medicine. In the Lindberg Lab we ...blogger Iris Lindberg, Professor at the University of Maryland School of Medicine. Iris Lindberg is a professor...grease, place and fill a dozen cloning rings, the remainder of the colonies on the plate dried out and died... cell clones with sterile truncated pipet tips. Indeed, an experienced operator can pick 48 clones from...mm dish ready for picking is shown in figure 1.   Find Additional Useful Protocols on Addgene's Protocol...antibiotic-resistant colonies, use a Sharpie to circle 12-48 individual colonies by (carefully) holding plates up to ...identified from the plate circling- are then individually punched out, gently sucked up into the pipettor...

  16. Interview: Hodaka Fujii on enChIP, New CRISPR Tools, and More

    Type
    Blog Post
    Published
    Dec. 2, 2014, 7:23 p.m.
    ... DNA-binding molecules for the first time and named the new technology as engineered DNA-binding molecule-mediated...also developed the inducible translocation trap (ITT) system to analyze signal-induced nuclear translocation...immunoprecipitation (iChIP) and engineered DNA-binding molecule-mediated chromatin immunoprecipitation...insertion of recognition sequences of an exogenous DNA-binding protein such as LexA into the target locus, which...is the rate-limiting step. When engineered DNA-binding molecule technologies such as TAL proteins and ...molecules makes it much easier to knock-in LexA binding elements into the target region. However, it struck...struck me that direct use of such engineered DNA-binding molecules even enables us to skip the step of knocking-in...

  17. Hot Plasmids - June 2020 - Barcoded CRISPR Library, Sparse Cell Labeling, Calcineurin Reporter, and DNA Staining Dye Alternative

    Type
    Blog Post
    Published
    June 2, 2020, 1:15 p.m.
    ...stained with PI (pink) to indicate cell death. Image from Diessl et al., 2020. Find the GFP reporter calcineurin...deposits and we hope they'll make it easier for you to find and use the plasmids you need. If you'd ever like... for a low number of cells and in vivo settings. Find the sgRNAiBAR library!  Zhu et al., Genome Biology...cells in a population, it’s easier to visualize individual/non-overlapping cells. Until now, tools for doing...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ...dephosphorylates the protein CZI, which in turn binds to calcineurin-dependent response elements (CDREs...fusions with tTALE to “stain” DNA. TALE proteins can bind specific DNA sequences which brings the attached...

  18. HA Frankenbody, a New Imaging Tool to Visualize Single Molecules and Nascent Peptides

    Type
    Blog Post
    Published
    Oct. 10, 2019, 12:41 p.m.
    ... fluorescent protein guide Find plasmids for subcellular localization Find fluorescent protein plasmids...Part stable scaffold, part epitope binding region, all fused to a fluorescent protein. “Frankenbody,”...antibodies for tagging purposes. Different antibodies bind different protein sequences called epitopes and ...interest. Antibody based probes, such as antigen-binding fragments (Fabs) (Porter 1959) and single-chain... 1972) are fused to mature fluorophores, and can bind and light up epitope tagged proteins in vivo. These...CDRs, or loops) of an antibody that recognize and bind specific epitopes (Ewert et al., 2004). This scFv...to easily observe the tagged protein upon probe binding in living cells.  In their proof of concept experiment...

  19. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    Published
    Sept. 17, 2024, 1:15 p.m.
    ...Antibody not completely binding Increase incubation time Not enough antibody binding sites Polyclonal...the technical design of your blot, but are still finding yourself having issues, you’re in the right place...compartment. If your proteins are nuclear- or DNA-binding proteins, you may need to sonicate your lysates...lysates in order to release the proteins from their binding. If you’re seeing protein aggregation, you may ...or improperly made running buffers can cause all kinds of odd issues. If your gel is running properly but...ladders contain either ‌a protein tag or an IgG binding site, allowing you to visualize them with a conjugated...double-membrane transfer with a psi membrane stacked behind the p membrane. If your protein runs through the...

  20. pCXLE toolkit: Efficient episomal plasmid-based method to reprogram peripheral blood cells to iPSCs

    Type
    Blog Post
    Published
    Dec. 14, 2017, 2:08 p.m.
    ...possible to reprogram differentiated cells into induced pluripotent stem cells (iPSCs) by the addition ...perform cell therapy and to model diseases in vitro. Find stem cell plasmids at Addgene Many sources of adult...and the EBNA-1 sequence that encodes for the DNA binding protein EBNA-1 [7]. These two sequences are necessary...cells [8]. The EBNA-1 protein recognizes OriP and induces amplification of the episomes concurrently with...such low efficiency of reprogramming is a major hindrance in using PBMCs as a source of somatic cells for...Cukier, and Derek M. Dykxhoorn. "Blood Derived Induced Pluripotent Stem Cells (iPSCs): Benefits, Challenges...PMC5118044. 2. Kim, Y., et al., The Generation of Human Induced Pluripotent Stem Cells from Blood Cells: An Efficient...

  21. The PAM Requirement and Expanding CRISPR Beyond SpCas9

    Type
    Blog Post
    Published
    Aug. 20, 2020, 2:30 p.m.
    ...). The 3-5 nucleotide PAM sequence serves as a binding signal for Cas9 and this sequence is a strict requirement...identified four novel SpCas9 variants with altered PAM binding specificity: SpCas9 variant Mutations (relative...VRER variants is similar to wild-type SpCas9, indicating that the variants are likely just as selective...sequence may not be characterized Non-SpCas9's bind a variety of PAM sequences, which makes them useful...550:280–284 . https://doi.org/10.1038/nature24049 Find plasmids from this paper at Addgene. Amrani N, ...–1027 . https://doi.org/10.1126/science.aaq0180 Find plasmids from this paper at Addgene. Kim D, Kim...Biotechnol 34:863–868 . https://doi.org/10.1038/nbt.3609 Find plasmids from this publication at Addgene. Kim ...

  22. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    Published
    April 13, 2021, 1:15 p.m.
    ... self-curing, an arabinose inducible lambda-red gene, and an IPTG-inducible sgRNA sequence targeting the...from our CRISPR topic overview Find other CRISPR methods for bacteria Find all articles about microbiology...editing, pTargetF can be cured from the cell by inducing expression of the sgRNA that targets the pMB1 ...updated pEcCas/pEcgRNA system, and confirmed that indeed the gRNA-pMB1 had a slightly higher leaky expression... includes versions of plasmids with different induction systems, such as PT7, Prha, Ptet, and Para. The...that is 2.6 times higher when compared to the industrial strain. In our metabolic engineering case study...high-yielding strain which had 6 times the yield of the industrial strain. Not only is MUCICAT better, it is faster...

  23. Rett Syndrome: A History of Research and Therapeutic Outlooks

    Type
    Blog Post
    Published
    Nov. 19, 2020, 2:15 p.m.
    ...editing or the induction of iPSCs, are available in the Resource Center. Researchers can also find links to ...so that both veteran and new Rett researchers can find the reagents and resources they need to cure Rett...Syndrome? Caused by a mutation in the methyl-CpG binding protein 2 (MECP2) gene, Rett Syndrome is a rare...normal development through ages 6 to 18 months, individuals with Rett syndrome experience regression of previously...Syndrome (Amir et al., 1999). We now know that MECP2 binds to methylated CpG dinucleotides and CAC trinucleotides...excision of the Stop cassette using tamoxifen-inducible Cre not only halted the neurological damage but...regardless of age or symptom severity. This exciting finding injected hope that a similar reversal may be possible...

  24. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
    Published
    June 28, 2016, 10:59 a.m.
    ... Resources on Addgene.org Find Software Tools to Help Design You gRNA Find CRISPR Plasmids for Use in... probably got a gene you’d like to manipulate in mind. Now it’s time to consider where you would like ...DNA supply company you currently use. You can also find many previously validated gRNAs at Addgene. In vitro...control, 1; very dark). Brighter fluorescence indicates a more successful gRNA design. Adapted from Mashiko...once they have been generated (more on that later). Find Validated gRNAs to Target Mouse Genes To begin the...plasmid using standard cloning techniques (The XX indicates where your target is inserted, disrupting the ...your modified pCAG-EGXXFP plasmid along with the individual gRNAs in their PX330 plasmids (or similar plasmids...

  25. Hot Plasmids - January 2023

    Type
    Blog Post
    Published
    Jan. 31, 2023, 2:15 p.m.
    ...benefit from region-specific, inducible expression.   Find heat-inducible lentiviral plasmids here!   Wu...what you'll find in this post: CRISPR-Csm: A novel RNA targeting system Heat-inducible lentiviral Car...Adapted from Figure 3 Colognori et al 2022.   Find CRISPR Csm plasmids here! Colognori D, Trinidad ...Epub ahead of print. PMID: 36690762.   Heat-inducible lentiviral CAR constructs by: Brian O'Neill  A...lab.  The crux of the system is a set of heat-inducible lentiviral CAR constructs, the most basic of which... which is depicted here: The heat-inducible Hsp (heatshock promoter) drives expression of the tumor-... signal in immunoassays due to only having one binding site per protein. Having multiple antibodies allows...

  26. Visualizing Translation at the Single Molecule Level

    Type
    Blog Post
    Published
    Aug. 1, 2017, 1:15 p.m.
    ...ribosome moves along an mRNA, it displaces other RNA-binding proteins to allow translation to occur. For a TRICK...requires the following components: a GFP protein that binds to the coding region of a reporter mRNA via a PP7... coat protein (NLS-PCP-GFP) an RFP protein that binds to the 3’ UTR of the reporter mRNA via an MS2 coat... the fluorescent proteins and is driven by an inducible promoter (Plasmid 64542 or 64543) Before translation...signal, NLS-PCP-GFP then returns to the nucleus to bind cognate untranslated mRNAs. The high specificity...multiple copies of the PP7 and MS2 coat protein binding sites to tether many copies of the FP-coat protein...development, the osk-TRICK reporter was double labeled, indicating translational repression. During later stages...

  27. Technical Design of a Western Blot

    Type
    Blog Post
    Published
    Aug. 22, 2024, 1:15 p.m.
    ...assay run time to two days.  Indirect or direct detection method Indirect detection is the most common...quicker to use, with high binding capacity, though it has lower retention after binding and washes. It’s less...for validation data, or follow the references to find the original publication and ensure that it includes...for a western blot. Polyclonal antibodies, which bind to multiple sites on an antigen, are cheap and can...the manufacturer, each rAb in a mixture can be individually validated. ‌rAbs are not as commonly available...). Direct westerns are simpler and faster than indirect ones and can be used in highly quantitative assays...tested to ensure it doesn’t impact the antibody’s binding to your protein of interest. Protein preparation...

  28. What's New in CRISPR - Winter 2018

    Type
    Blog Post
    Published
    Dec. 18, 2018, 1:17 p.m.
    ...factor localization and binding via ChiP-seq. Learn more about ENCODE Find the transcription factor...Addgene.org Read our CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ... but we hope that this blog series will help you find new CRISPR tools for your research.     Improved...sensitivity, and more. Read the paper in Science Find the new SHERLOCK plasmids More precision with ...Cas9’s. Read the paper in Nature Communications Find the Cas9-Cas9 chimera plasmids A modular toolkit...engineering in plants. Read the paper in bioRxiv Find the plant genome editing toolkit New Cas protein... Addgene blog post Read the paper in Science Find the Cas14 plasmids Anti-CRISPR proteins for optogenetic...

  29. Special Delivery: Fluorophore Targeting for FRET Studies

    Type
    Blog Post
    Published
    July 19, 2016, 2:30 p.m.
    ...substrate or add an essential cofactor? How many binding partners does it have and how do they come off ...package needs to go to the proper mailbox (i.e., a binding site) in the right city (protein). In a cell, this...thousands of biomolecules each containing multiple binding crevices that can nonspecifically soak up your ...when donor and acceptor are close together. The binding of acceptors >100 Å away from the donor doesn’t...translation, the novel tRNA/unnatural amino acid finds the UAG codon in the ribosome, resulting in site-specific... reagents You may already have a FRET acceptor-binding site on your protein of interest: you just don’...NTA-sepharose beads used for protein purification: the NTA binds to the His tag on your protein and the coupled Cy...

  30. Hot Plasmids: Summer 2024

    Type
    Blog Post
    Published
    Aug. 27, 2024, 1:15 p.m.
    ...specified by the DNA-binding protein. The ADD domain of the methyltransferase binds to H3K4me0 on D3L, ... our Hot Plasmids articles. Here's what you'll find in this post: MagIC-Cryo-EM for enrichment and ...could be used for applications beyond cryo-EM.   Find plasmids for MagIC-Cryo-EM here!   Arimura, Y., ...samples from damage at the AWI. Plus, tardigrades!  Find RvLEAMshort here!    Abe, K. M., & Lim, C. J. (2024... to partially correct mouse models of genetic blindness (An et al., 2024) — the first demonstrated use... a genetic disease phenotype in an animal model. Find PE-eVLP plasmids here! The lab’s other recent deposit...four-fold improvement over the wild-type enzyme. Find optimized PASSIGE plasmids here!     Figure...

  31. Plasmids 101: Biotinylation

    Type
    Blog Post
    Published
    Nov. 15, 2018, 1:50 p.m.
    ...while its binding partner, avidin, is found in the egg white. Avidin is a glycoprotein that can bind four ...to your sample. Any free biotin-binding sites on this complex can bind to your biotinylated antibody. ...Biotin and its binding partner avidin are commonly used today in molecular biology for an array of different...was able to purify the protein responsible for binding biotin, avidin. Avidin was aptly named for the ...purified from. Snell later confirmed that avidin was indeed the culprit in egg white injury by feeding rats...consists of four identical subunits that can each bind one molecule of biotin. Biotin and avidin have an... reactive species without affecting the avidin binding site. With all of these features it isn’t surprising...

  32. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    Published
    Sept. 5, 2017, 1:58 p.m.
    ...RNA binding site works well. However, since some guide RNAs may tolerate a mismatch in the binding sequence...purpose of making sequence-specific changes in human induced pluripotent stem cells (iPSCs). The CRISPR/Cas9... dele­tions or other mutations, referred to as ‘indels’. While this is convenient for generating gene ... intended HDR events were corrupted by unwanted indels on the same allele, probably due to concomitant...combined pitfalls made it virtually impossible to find the 0.2-0.5% correctly edited alleles, when screening...accurately edited, the other likely contains unwanted indels. Following this observation, we tested a strategy...bases as close to the PAM as possible (see below to find out why). Please check our recent studies for details...

  33. Thoughts on the Future of Research 2015 Boston Symposium

    Type
    Blog Post
    Published
    Nov. 25, 2015, 4 p.m.
    ...and research independence needed to pursue his or her chosen career path,” but individual institutions...scientifically minded, and able to communicate complex information - all traits you are likely to find in graduate...underrepresented individuals too nicely. When the work of the underrepresented individual is then judged...the FOR website, the FOR Boston 2015 organizers indicate that the main question they wanted to focus on... “How many graduate students/post-docs?” You can find some of the more detailed questions the organizers...The National Academies define a postdoc as “An individual who has received a doctoral degree (or equivalent...during the Saturday Hack Day Session focused on finding this information and making it more easily digestible...

  34. What's New in CRISPR - Spring 2019

    Type
    Blog Post
    Published
    March 21, 2019, 12:47 p.m.
    ...Addgene.org Read our CRISPR guide Find CRISPR plasmids by function Find recently deposited plasmids ... but we hope that this blog series will help you find new CRISPR tools for your research.     CRISPR-...sites. Read the paper in Nature Communications Find the Cas12b plasmids  Plant base editing with a... and wheat. Read the paper in Genome Biology Find plasmids containing these base editors Spatially...the paper in Frontiers in Molecular Neuroscience Find the AAV-based SaCas9 genome editing plasmids String...can all be chosen. Read the paper in PLoS One Find the STAgR cloning plasmids Mobile-CRISPRi allows...conjugation.   Read the paper in Nature Microbiology Find the Mobile-CRISPRi plasmids Identification of ...

  35. Viral Vectors 101: Calcium Sensors

    Type
    Blog Post
    Published
    Aug. 10, 2023, 1 p.m.
    ...encoded calcium indicators (GECIs) are fluorescent proteins linked to calcium binding proteins (e.g., ...nucleus-targeted GCaMP6 with an extra apoCaM-binding motif to prevent aberrant binding of the biosensor to endogenous...in living cells and organisms. Unlike chemical indicators, these DNA constructs can be delivered to intact...low affinity calcium biosensor, so that the (1) binding of calcium, (2) allosteric rearrangements of the...is engineered with a reduced number of calcium binding sites per sensor (to decrease unnecessary calcium...:509-18. Inoue M. Genetically encoded calcium indicators to probe complex brain circuit dynamics in vivo...Kanemaru K, Iino M. Genetically encoded fluorescent indicators for organellar calcium imaging. Biophysical journal...

  36. Educational Planarian Tools for Teaching Developmental Biology Techniques

    Type
    Blog Post
    Published
    May 24, 2018, 1:20 p.m.
    ...CRISPR Resources at Addgene.org Find plasmids from the Sánchez lab Find plasmids for other developmental... used for both in situ hybridization and dsRNA-induced RNAi experiments. Notably, this plasmid harbors... more detailed materials and protocol resources. Find Sánchez lab Plasmids Visualizing gene expression... example, Digoxigenin-labelled Uracil) that will bind to complementary mRNA within an embryo or tissue... expression of specific genes. After riboprobe binding (via hybridization) specific antibodies that detect...referred to as small interfering RNAs (siRNA) and they bind to a class of Argonaute proteins. The Argonaute ...complementarity to the remaining single stranded siRNA to bind natively expressed mRNAs.These native mRNAs are ...

  37. Phage Directory: From Phage Therapy to a Repository of Phage Information

    Type
    Blog Post
    Published
    Jan. 10, 2019, 2:38 p.m.
    ...quickly find much-needed phages for patients.   A phage directory is crucial because it's hard to find the... blog posts on open science Find blog posts about scientific sharing Find blog posts about networking ...Alberta, saw that scientists were using Twitter to find phages to treat an antibiotic resistant bacterial...designer that she knew. “We had actually met at a lindy hop swing dance event,” Zheng says (networking can...quickly teamed up to create Phage Directory in a whirlwind three days in November 2017. #Phage researchers...antibiotics. We need lytic non-lysogenic phage URGENTLY to find suitable phage matches. Email [email protected]... comes to administering phage therapy. The laws behind phage therapy, acceptable endotoxin levels, etc...

  38. AAV Vector Quality Control: Going the Extra Mile with NGS

    Type
    Blog Post
    Published
    Sept. 12, 2017, 1:44 p.m.
    ...prep. With this in mind, we recommend that users titrate their AAV vectors to find the optimal dose that...Quality Control Resources on Addgene.org Find Ready-to-Use AAV Find Ready-to-Use Lentivirus Browse Viral ...used to repeating experiments to confirm their findings. But no scientist wants to repeat an experiment...step process using Geneious software: First, the individual sequencing reads (~150 bp each) are aligned to... reviewed. As mentioned earlier, it is common to find DNA from the packaging cell genome, bacterial genomes...our pipeline (see Figure 2, sample 2). When we blindly analyzed AAV-68544, we were easily able to identify...Recombinase-dependent sequences may be subject to recombinase-independent recombination during plasmid growth in bacteria...

  39. Genetically-encoded Sparse Cell Labeling - A SPARC of Innovation

    Type
    Blog Post
    Published
    May 21, 2020, 1:15 p.m.
    ...transcription factor binding site. GAL4 expressed in the fly binds to the UAS binding motif, driving downstream...cells in a population, it’s easier to visualize individual/non-overlapping cells. While transgenic animals...require heat-shocking the system or using chemical inducers of gene expression (del Valle Rodríguez et al....controlled by the upstream recombination outcome. Find SPARC plasmids at Addgene To more precisely control...intermediate (d), and sparse (e) labeling of the calcium indicator, GCaMP6f, in the Drosophila optic lobe. Image ...organisms by replacing the transcription factor binding site and effector in the construct. In Drosophila...dendrites of T5 neurons by placing the calcium indicator GCaMP6f in the effector position, as well as to...

  40. Plasmids 101: Secondary Nanobody Toolbox

    Type
    Blog Post
    Published
    Feb. 27, 2018, 2:04 p.m.
    ... of the antibody has an antigen binding site for a total of 2 binding sites per antibody. C) IgG’s are...Advantages of Nanobodies How to Use the Nanobody Toolbox Find Nanobody Plasmids Structural comparison As the...Antibodies One of the main reasons antibodies are great indirect immunodetection tools is their strong signal strength...occur: A) Multiple polyclonal secondary IgGs can bind to different epitopes of a primary antibody. B) ...displacement 2° incubation can be skipped by pre-binding to 1° Monovalent Monoclonal Only anti- rabbit...IgG subclasses. Instead, you can pre-incubate individual primary antibodies with secondary nanobodies ... with three IgG1 antibodies. Antibodies were individually incubated with a secondary nanobody conjugated...
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