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We narrowed to 698 results for: des.1

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  1. Luciferase Plasmid Collection

    Type
    Collection
    ...applications. LuxSit-i : An artificial luciferase desined de novo using deep-learning approaches. Split-Luciferase... Eric Campeau 21474 pLenti CMV V5-LUC Blast (w567-1) Firefly CMV Lentiviral expression of firefly luciferase...to that of the fluorophore. Conformationally destabilized Firefly luciferase mutants : A set of three ...three sensors that are increasingly structurally destabilized versions of Firefly luciferase which serves ...dimerization , endosomal escape of cell-penetrating peptides , and more. nLuc/cLuc for split Firefly Luciferase...mammalian systems. ID Plasmid Luciferase Type(s) Description PI 16539 pBV-Luc Firefly Insertion of 5' promoter...service icon . ID Plasmid Luciferase Type Promoter Description PI 60226 AAV:ITR-U6-sgRNA(backbone)-pEFS-Rluc...

  2. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    ...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...bands, you may want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to make sure...Membranes look the same from both sides, so it’s quite easy to think lane 1 is‌ lane 8. Common ways to keep...reagent (usually an antibody).      Figure 1: A protein gradient. The density of the bands, which...

  3. Antibody Guide

    Type
    Collection
    ...and placed in a buffer. Antibody Structure Figure 1: Structure of an antibody A standard antibody is made... together to form a “Y” shape, as shown in Figure 1. The two arms of the Y structure are responsible for... is described in more detail in the Multiplex Assays section below. The following table describes the ... Science Guides Antibody Guide Antibody Guide View Addgene's Antibody...of B cells isolated from animals immunized as described above are used to create hybridomas, which produce...each antibody has two binding sites. Isotype Description Configuration and Valency Subclasses IgA Prevalent... used to quantify relative amounts of protein. Includes: Western blot ELISA Capture - used to isolate ...

  4. Typing CRISPR Systems

    Type
    Blog Post
    ...   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...effector was developed, called Cas7-11. Despite being in Class 1, Cas7-11 is a single protein effector ...types, 33 subtypes, and 17 variants. Figure 1: Overview of the CRISPR classification hierarchy. ...effector complex after binding to a target. Class 1 is the most abundant of the two, comprising about ...incredible abundance, Class 1 CRISPR systems are less popular in the lab. This can be attributed to the multi-...

  5. Antibodies 101: Normalization and Loading Controls for Western Blots

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

  6. The Effect of COVID-19 on Liu Lab Plasmid Requests From Addgene Data

    Type
    Blog Post
    ...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...are no exception to this effect -- meticulously designed experiments in labs around the world were put ...

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

    Type
    Blog Post
    ...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...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...

  8. Plasmids 101: Biotinylation

    Type
    Blog Post
    ...7.1 (2014): 1–7. PubMed PMID: 26224555. PubMed Central PMCID: PMC4884002. 10. Boer, E. de, Rodriguez, ...attach proteins or polypeptides to a solid substrate (Holden and Cremer, 2003). References 1. Chapman-Smith... of the biotin-avidin interaction   Figure 1: Biotin is enriched in egg yolk while its binding ...matrices.” Journal of Nanobiotechnology 13.1 (2015): 1–14. PubMed PMID: 25980573. PubMed Central PMCID: PMC4461904...Northern blotting Cell surface labelling Attaching polypeptides to a substrate Flow cytometry/fluorescence-activated...your downstream application. Biotin labelled oligonucleotides can be used in a multitude of applications...

  9. MXS Chaining

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

  10. Plasmids 101: Dimers and Multimers

    Type
    Blog Post
    ...indicates the presence of a multimer.   Fig. 1: The monomer of Plasmid 12259: pMD2.G (depicted on...plasmid sequencing services using long-read sequencing (1-25 kb), such as Oxford Nanopore sequencing or PacBio...10017-26. doi: 10.1074/jbc.M110.176636. Epub 2011 Feb 1. PMID: 21285356; PMCID: PMC3060453. Bazaral M, Helinski...multiple circular DNA forms of colicinogenic factor E-1 from Proteus mirabilis. Biochemistry. 1968 Oct;7(10...Strausbaugh LD, Gray P, Vartak NB, Brown M, Talbot D, Berg DE. pBR322-derived multicopy plasmids harboring large...Methods Mol Biol. 2009;582:11-25. doi: 10.1007/978-1-60761-340-4_2. PMID: 19763938. Maucksch C, Bohla A...confirmations like linear and nicked plasmids, as described in this article.   Long-read sequencing Addgene...

  11. X-CHIME: Context Dependent Germline Knockout in Immune Cells

    Type
    Blog Post
    ...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, et al. 2024.   The X-CHIME deposit also includes pXPR_071, a plasmid that leads to higher titer...blog CRISPR101: Validating Your Genome Edit How to Design Your gRNA for CRISPR Genome Editing CRISPR Cheat...

  12. Deep Mutational Scanning with One Pot Saturation Mutagenesis

    Type
    Blog Post
    ... mutagenic primers to introduce all desired codon changes (step 1 in the image above). A second universal...Supplementary Note 1 in Wrenbeck et al for details on how nicking was optimized. How should I design my mutagenic...making some or all of the following modifications: 1) increased concentration of Taq polymerase, 2) increased...introduced, and scalability (see Supplementary Table 1 of Wrenbeck et al). It uses a uracil labeled wild-...strand is nicked by the BbvCI variant not used in step 1. Then this strand is degraded with ExoIII and ExoI... the first digest (step 1 in above diagram). Remember that the BbvCI enzymes nick the DNA strand that ...time no, but there are 2 instances where it does. 1) When the backbone vector has multiple BbvCI sites...

  13. Important Considerations When Using AAVs

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

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

    Type
    Blog Post
    ...HA-tagged proteins in vivo. Nature Communications, 10(1). PubMed PMID: 31270320. PubMed Central PMCID: PMC6610143...epitopes (Ewert et al., 2004). This scFv hybrid provides the probe structure and stability from the scaffold...translated quickly by the ribosome. This feature provides HA frankenbody the ability to bind nascent HA ...

  15. Antibody Validation for Flow Cytometry

    Type
    Blog Post
    ...workshops. Let’s use TIM-1 (Single pass type-1 membrane protein), which has been designated CD365 in the most...dye, respectively. WT and KO cells were mixed in a 1:1 ratio and fixed in 4% PFA and permeabilized in 0.1%...antibodies were diluted to 1 µg/mL except for 14558, which was used at 0.35 µg/mL (1/100). * = monoclonal antibody...specific antibody:target combination of interest (Figure 1). It can therefore be helpful to try all three when...to label an intracellular target.   Figure 1: HCT 116 WT and SYT1 KO cells were labelled with a...cells were stained with the indicated Synaptotagmin-1 antibodies and corresponding Multi-rAb CoraLite® Plus...between the wild-type and the KO population (Figure 1), further optimization can be performed to both maximize...

  16. When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

    Type
    Blog Post
    ...these situations in cells are described below (See Figure 1).   Figure 1: Each problem with dimerizing... in nature are prone to dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [...dimerizing fluorescent proteins is illustrated. 1) Membrane proteins fused to FPs that dimerize can form dynamic...active state due to closure by FP dimerization.   1. Transmembrane fluorescent protein fusions Transmembrane...bright, non-physiological, and difficult to ignore [1, 2]. 2. Fusion to an obligate dimer or oligomer Several... For a more detailed discussion see [5]. Problems 1 and 2 depend on protein concentrations. An imperfect... distance runner, gardner, and cook.   References 1. Snapp, E.L., et al., Formation of stacked ER cisternae...

  17. Predicting Adverse Reactions to Monoclonal Antibody Drugs

    Type
    Blog Post
    ...immunogenicity score (Mattei et.al., 2022).    Figure 1: Generation of immunogenicity scores by predictive...Terry, F. E., Roberts, B. J., Rosenberg, A. S., & De Groot, A. S. (2022). In silico Immunogenicity Assessment...antibodies for the treatment of diseases. J Biomed Sci 27, 1 (2020). https://doi.org/10.1186/s12929-019-0592-z  ...in vivo animal models. In these cases, they are designed to mimic the human lymphatic system or even the....org/10.3389/fddsv.2022.952326Joubert, M. K., Deshpande, M., Yang, J., Reynolds, H., Bryson, C., Fogg,...

  18. Degrading DNA with Cascade-Cas3

    Type
    Blog Post
    ...complexity. Figure 1: Schematic of Class 1 CRISPR types. Class 1 includes type I with a Cas3 effector...collection! A “complex” system Cas3 is part of the Class 1 CRISPR family, which means it requires multiple Cas...the brunt of the mechanical work for editing. Class 1 CRISPR systems make up the majority of CRISPR-based...CRISPR-based immune systems in bacteria and archaea. Class 1 is further broken down into different types, with ...making up type I, the most abundant type in the Class 1 category. These type Is can then be further broken...editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-13226-x Cameron...Cas11. Type I-E Cascades contains all five of these Cas proteins, while type I-C Cascades do not contain...

  19. Save Time with Transient Plant Leaf Transformations

    Type
    Blog Post
    ... medicinal plant, the Catharanthus roseus (Figure 1). The ultimate goal of our research is to enhance ... which one you choose depends on your experiment (de Ruijter et al., 2003). For example, we did our initial...Humana Press, 2009. 41-50. PubMed PMID: 19183892. De Ruijter, N. C. A., et al. "Evaluation and comparison...activities in seed plants." Plant cell reports 9.1 (1990): 1-5. PubMed PMID: 24226366. Lee, Min Woo, and Yinong...and a vacuum is applied with a vacuum bell or desiccator. Successful infiltration can often be seen by...

  20. Mouse Modeling, Part 2: Breeding and Crossing Mice

    Type
    Blog Post
    ...approximately 1 in 16 mice that harbor homozygous alleles at both of your genes of interest. About 1 in 4 mice...In Part 1 of our mouse modeling blog series, we covered techniques that can be used to introduce genetic...mice in your experiment. As you’ve learned in Part 1, there are many types of genetically engineered mice..., and assuming Mendalian patterns of inheritance: 1. Genotype your parental mice In order to ensure that...example of mice that are derived from crosses. In Part 1, we explained how genetic recombination in mice expressing...expressed, or inducible (this is explained more in Part 1 of this series). In any case, Cre is a dominant phenotype... allele decreases. Crossing mice to generate a desired phenotype Instead, genetic engineering should be...

  21. CRISPR-mediated Plant Base Editors

    Type
    Blog Post
    ...increases the generation of the desired T-A base pair. Figure 1: Transition mutation (C>T, G>A,...excision repair mediated by uracil glycosylase (Figure 1). The presence of a uracil glycosylase inhibitor (... A-T base pair is converted to a G-C pair (Figure 1). As the cellular inosine excision repair is not as...Target-AID based (CBE-2)-  and rAPOBEC based (CBE-1)-  CBEs are successful base editors in rice, tomato...deaminase can edit in a stretched window ranging from 1-17 bp relative to the PAM sequence (Zong et al., 2018...which recognize PAM sequences other than NGG (Table-1). Type Nickase Cas9 (D10A) Deaminase PAM requirements...locus/loci, choose a base editor variant from Table 1 depending on the PAM sequence. Avoid targeting a ‘...

  22. Bricking Science: Portraying Scientific Reality Through LEGO

    Type
    Blog Post
    ...thanks to our guest blogger, Dalila Cunha de Oliveira. Dalila Cunha de Oliveira is a PhD student in Hematology...post was contributed by guest blogger, Dalila Cunha de Oliveira. Bricking Science is an idea built, literally...motion videos is very time consuming; producing a 1-2 minute video takes an entire week of our free time...with my husband, I thought about how the limitless design possibilities of the LEGOs were mirrored by the...made, colorful laboratory. When we started, the hardest part of the process was to embed personality in...

  23. Cancer, Inflammation and Immunity - Harnessing the Body’s Defenses to Fight Cancer

    Type
    Blog Post
    ...aggregation, and potentiates the effect of PD-1 blockade.“ (Ref 4). PD-1 helps tumor cells evade the immune system...a positive effect on cancer treatment (Ref 1). As described in the presentation, starvation or the use...of the immunosuppressive cell surface receptor PD-1. This immunosuppressive environment in turn may represent...  (Chimeric Antigen Receptors) CAR T cells and PD-1 modulators. An exciting development in immunotherapy...free deposit and distribution service! References: 1. Pietrocola, Federico, et al. "Caloric restriction...field of Immuno-Oncology, a sizzling hot topic, includes studies on novel cancer therapeutics such as  ... with T cell receptor engineering (Ref 5)  as described by Blue Flame Addgene depositor Professor Michel...

  24. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    ...can use Figure 1 to compare the genomes of these two family members.   Figure 1: Gamma-retrovirus...Retroviruses” is an umbrella term which encompasses 1.) the gamma-retroviruses — that have been developed...of retroviruses called the lentiviruses (like HIV-1) — that have been developed into lentiviral vectors...Academy of Sciences of the United States of America, 54(1), 137–144. https://doi.org/10.1073/PNAS.54.1.137. ...1997). Functional RT and IN incorporated into HIV-1 particles independently of the Gag/Pol precursor protein...RNA as template. Journal of Molecular Biology, 79(1), 163–183. https://doi.org/10.1016/0022-2836(73)90277...Aids Full-Length Human Immunodeficiency Virus Type 1 DNA Synthesis. Journal of Virology, 82(5), 2376. https...

  25. Hot Plasmids February 2024

    Type
    Blog Post
    ...Biotechnology, 10.1038/s41587-023-01756-1. https://doi.org/10.1038/s41587-023-01756-1. PMID: 37095348. Breaking...minute incubation, even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused to HIV...expressing COX8a-mStayGold. Scale bars 10 μm (main), 1 μm (inset). Image reused from Ando et al. 2023, under... membrane-targeting applications. Nature Methods, 1–9. https://doi.org/10.1038/s41592-023-02085-6. PMID...StayGold fluorescent protein. Nature Biotechnology, 1–4. https://doi.org/10.1038/s41587-023-02018-w. PMID...broad tropism in primates. Nature Communications, 14(1), 3345. https://doi.org/10.1038/s41467-023-38582-7... They fused the Cas enzyme to cell-penetrating peptides and co-incubate it with an “assist peptide” that...

  26. An “elegans” Approach to Better CRISPR/Cas9 Editing Efficiency

    Type
    Blog Post
    ...perform pha-1 co-conversion are available through Addgene. In 2019, Farboud et al. described another similar...effective recovery of knock-ins, but did result in 1-2 kilobases of additional sequence being introduced...facilitating recovery of edited animals. Figure 1: In the background are 504 restriction digests of ...selection for repair of a temperature-sensitive pha-1 mutation significantly enriches for knock-in of 2x... 3xFLAG epitopes into other, non-linked loci; pha-1(e2123) mutant worms are perfectly viable at 15 ºC,...https://doi.org/10.1093/nar/gku289 Chen C, Fenk LA, de Bono M (2013) Efficient genome editing in Caenorhabditis... DNA double-strand breaks up to 54 bp from the desired insertion site result in efficient editing. Repair...

  27. Addgene Depositors Get More Citations

    Type
    Blog Post
    ...of publication. Table 1 provides an example calculation of the weights. Table 1: Journal Weighting Example...amassed nearly 2,400 citations in its first four years (1). In addition to publishing in Science, Professor ...papers without plasmids deposited with Addgene (Figure 1). Even aggregated this much, you can see the 2013...(F) Weight (=F/E) (G) Weighted Citations (=C*G) 1 2010 3 Nature  3.3% 10.5%   3.2  9.6 2 2010...website at: http://www.neil-t.com/ .   References 1. Cong, Le, et al. "Multiplex genome engineering using... Science. For each publication, Web of Science provides basic bibliographic information and uses its vast...

  28. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    ...which is also gaining a lot of new traction lately (1, 2), depends on knowledge of the structure(s) a given...RBPs successfully identified using this method. Step 1: Prepare a random fragment library of phage DNA This...first step involves digesting phage genomic DNA into 1-3 kb (approximately gene-sized) fragments so that ...in the bulk of resultant fragments falling between 1 and 3 kb results in optimal likelihood of capturing...solution-soaked filter paper (colony-side up) and leave for 1 h at room temperature. After this, it is helpful to...proteins expressed by your library. After blocking for 1 h at room temperature with gentle shaking, gently ... to incubate the membranes overnight in 500 mM to 1 M NaCl in order to enhance folding of the recombinant...

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

    Type
    Blog Post
    ... Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously. Image from Li et...coli K-12 strains, 2 E. coli B strains, 1 E. coli W strain, and 1 Tatumella citrea, with a total of 70 sites... update the pCas/pTargetF system.   Figure 1: The editing process begins with the addition of the...updates involved the following changes. In pCas9, we 1) replaced the promoter of gRNA-pMB1 with PrhaB, 2)...biosynthesis, we constructed a strain library with 1-11 copies of integrated genes. We obtained a stable...CRISPR-associated transposases” or MUCICAT. Here, we’ll describe each of these CRISPR systems. The original pCas...by sacB counterselection.  The MUCICAT toolkit includes versions of plasmids with different induction ...

  30. Plasmids 101: Repressible Promoters

    Type
    Blog Post
    ...Claude Desplan. “Power tools for gene expression and clonal analysis in Drosophila.” Nat Methods 9(1) (2011...Bacillus. Appl Microbiol Biotechnol. 2019 Jan;103(1):303-313. doi: 10.1007/s00253-018-9485-4. Epub 2018...Methods Mol Biol. 2016;1478:53-78. doi: 10.1007/978-1-4939-6371-3_3. PMID: 27730575; PMCID: PMC5270762. ...2022). https://doi.org/10.1038/s41598-022-07852-7 De Boer, Herman A., Lisa K. Comstock & Mark Vasser. ... and lac promoters.” Proc Natl Acad Sci U S A. 80(1) (1983): 21-5. PubMed PMID: 6337371. PubMed Central...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...

  31. Adenoviral Vector Production and Troubleshooting

    Type
    Blog Post
    ...repeats (ITR) present at both ends.   Figure 1: A) The structure of an adenovirus: the virion is ... 5 key steps: Construction of the rAdV plasmid (~1 week): The AdEasyTM system is the most popular method...Addgene’s Adenoviral Guide webpage, and references 1, 2.*Pro-Tip*: once the correct recombinant pAdV plasmid...transfected with the AdV plasmid construct from step #1 and allowed to stay in culture for up to 20 additional... it will result in very low titer. Amplification (1-2 weeks) - The rAdV-S is used to infect more HEK293...-4 rounds) at increasing scale over the course of 1-2 weeks. Each round of amplification should result...CsCl are commercially available. Titer calculation (1 or 10 days) Physical titer (particle count, rAdV ...

  32. Live and Let Dye: Self-Labeling Protein Tags

    Type
    Blog Post
    ...delivery by modular envelope design. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467...intracellular targets. Nature Communications, 7(1), Article 1. https://doi.org/10.1038/ncomms10778 Erdmann...molecules in vivo. Nature Biotechnology, 21(1), Article 1. https://doi.org/10.1038/nbt765 Lavis, L. (2016... capping protein. Nature Communications, 14(1), Article 1. https://doi.org/10.1038/s41467-023-39655-3 ...up linked to whatever molecule you choose (Figure 1). Just express the fusion protein and add a specific...proteins, opening up new applications.   Figure 1: Live-cell labeling strategies. The green structure...minimally-disruptive genetic tag on the protein of interest. Table 1: Summary of popular self-labeling tag systems ...

  33. 22 Hot Plasmid Technologies from 2014

    Type
    Blog Post
    ...bacterial mutagenesis or gene targeting Victor de Lorenzo's lab has engineered a modular mini-Tn5 vector...on channelrhodopsins – Chronos & Chrimson Through de novo sequencing of 127 algal transcriptomes, as well...expression. Brown et al., Genesis 2014 Jan;52(1):39-48. APEX2 for proteomic mapping and electron...engineering cells that lack release factor one (RF-1; Bacterial strain EcAR7) and minimizing the set of... of human mitogen-activated ERK activating kinase 1 (MEK1) with either one or two phosphoserine residues...comprised of three sets of cloning vectors (Level 0, 1, or 2) which can be utilized in three successive assembly... vectors are directionally assembled into a Level 1 vector creating a single transcriptional unit (Ex:...

  34. The Breast Cancer Microenvironment: A Tumor’s Backstage Team

    Type
    Blog Post
    ...enzyme (among others): caveolin 1, also known as CAV 1. Usually, caveolin 1 promotes the cell life cycle ...caveolin 1, there’s less malignancy because there’s less proliferation. However, the lack of caveolin 1 in ...believes that miRNA may decrease the expression of TAK 1, an enzyme that controls apoptosis and regulates transcription...Agnieszka K., et al. "An absence of stromal caveolin-1 expression predicts early tumor recurrence and poor...to the development and spread of cancer cells. Besides these stromal cell modifications, stromal cells...

  35. Adenovirus Guide

    Type
    Guide
    ...to an antigen. AdEasier-1 cells BJ5183 E. coli cells containing the pAdEasy-1 packaging plasmid. Adenovirus...Oncolytic Referring to the destruction of cancer cells, generally by lysis. pAdEasy-1 Adenoviral backbone plasmid...icosahedral capsids and build new virions. Figure 1: Wild-type adenovirus and its genome. Genes over or... This construct is then transformed into AdEasier-1 cells (Addgene #16399) , which are BJ5183 E. coli ... viruses. The adenoviral backbone plasmid pAdEasy-1 (Addgene #16400) is suitable for most purposes. For...production system. Figure is based on the use of pAdEasy-1. Created with BioRender.com. AdMax™ AdMax™ is another...landscape . Signal Transduction and Targeted Therapy, 6 (1), 53. https://doi.org/10.1038/s41392-021-00487-6 (...

  36. Quickest Way to Deposit Plasmids: The Deposit Spreadsheet

    Type
    Blog Post
    ...red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have drop-down options for you...Table 1: Guidelines for the data fields in the deposit spreadsheet Data Field Description Plasmid...how to use our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — our tech transfer...scientist, you will have the most involvement with Step 1, and, while data entry can be a chore, we’ve made ...Deposit” and “Download Deposit Spreadsheet” (see Figure 1). Please read our “How to Deposit Your Plasmids with...thorough guide to our online data entry process. Figure 1: Begin the deposit process by clicking “Submit Online...lab. Plasmid Type Choose from: Encodes one insert, encodes gRNA/shRNA, or empty backbone. Purpose...

  37. Scientific Reproducibility - Focusing on Solutions at the Minisymposium on Reproducibility

    Type
    Blog Post
    ... handed out at the event). Program 0 - 1:24 - Intro 1:24 - 29:31 - Reproducibility Overview - Jeffrey... 49:45 0 1:18:45 - Reagent Development - Steven C. Almo, Institute for Protein Innovation  1:18:45 - 2...reproducibility As Jeffrey Flier points out in his talk (1:24 - 29:31), there are a variety of ways of defining...enterprise towards greater reproducibility. Cause 1: Inability to repeat previously performed experiments...the Institute for Protein Innovation (IPI) (49:45 - 1:18:45), there are also initiatives to create more ...found. Consider the hypothetical situation in Figure 1 where many scientists are performing similar experiments...experimental designs and we need to be transparent about the reasons we alter these designs if we do so...

  38. Supporting Rare Disease Research with BeHeard 2017

    Type
    Blog Post
    ...estimated to affect anywhere from 1 in 6,000 to 1 in 19,000 live births (1).  While disruption of the smn1... funding opportunities. References 1. Jones, C., et al. "PP09. 1–2352: Systematic review of incidence...rare diseases. These underrepresented individuals deserve a voice. Check back with Addgene often to learn...

  39. REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

    Type
    Blog Post
    ...deletions, point mutations PCR Reactions 1 1 1 per fragment 1 per fragment In vitro Assembly No No Yes...methods for site directed mutagenesis   References 1. Trehan, Ashutosh, et al. (2016). “REPLACR-mutagenesis...discussed methods. As described above, you design two primers, each containing the desired mutations, to amplify...make a point mutation, you design two oligonucleotide primers with the desired mutation, then use those ... figure above shows guidelines for primer design; a desired insertion should be present in both primers...vitro recombination. Across all modifications described in Trehan et al., REPLACR-mutagenesis’ median ...

  40. New Neuroscience Tool: The iGluSnFR3 Glutamate Sensor

    Type
    Blog Post
    ...sensing glutamate using both 1-photon and 2-photon imaging.   Figure 1: 1P and 2P excitation and ...hSynap: Human Synapsin-1 promoter. Good for neuronal expression. hSynap-FLEX: Synapsin-1 promoter, FLEXed for... v857. Image adapted from Aggarwal et al., Figure 1   Increased versatility through two different...expression in Cre-expressing cells. hSynap-FLP: Synapsin-1 promoter, FLP-dependent expression GFAP: Glial fibrillary...characterization of the new variants is available here. Upgrades to iGluSnFR3 To improve upon the SF-iGluSnFR, ...

  41. Fluorescent Tagging of Endogenous Genes with SapTrap

    Type
    Blog Post
    ...template (Fig. 1, Step 1). There is no need for PCR or cloning, as digestion of the destination vector with...selectable marker, and connector to be released (Fig. 1, Step 2-3). Since donor plasmids of the same type ...correctly assembles the final targeting vector (Fig. 1, Step 4), and co-injection of the targeting vector...human cells. To use CRISPaint, users need 3 vectors:  1) a gRNA vector that targets the gene of interest, ... Tagging with Short Homology Donors.” Cell Rep.15(1) (2016):210-8. PubMed PMID: 27052166. Kunzelmann,...SapTrap, the user first designs either oligos or synthetic DNA for the desired gRNA target sequence, as...specified by a guide RNA (gRNA) sequence. Users can design gRNAs to induce the break at a specific genomic...

  42. Plasmids 101: Gateway Cloning

    Type
    Blog Post
    ...pLenti CMV Puro DEST (w118-1) or the doxycycline-inducible pLIX_403. The chosen attR destination vector will... our desired constructs: lentiviral expression of the human KRAS gene in mammalian cells. STEP 1: Generate...recombination sites (attL-left- and attR-right-, Figure 1). Under certain conditions, the attL and attR sites...regeneration of attP and attB sites.   Figure 1: Lambda phage integration and excision reactions. ...the generation of the expression construct in only 1 day, as opposed to 2+ days with traditional restriction... Find Gateway cloning plasmids here! References 1. Chee JY, Chin CF (2015) Gateway Cloning Technology...clone in their desired DNA sequences. Gateway technology relies on the two reactions described below: The ...

  43. High Complexity Golden Gate Assembly with NEB’s DAD

    Type
    Blog Post
    ...reactions. (Need a refresher? The five rules are: (1) don’t use the same overhang twice; (2) avoid palindromes...Gate) pot (Pryor et al., 2022).     Figure 1: Workflow for high complexity Golden Gate Assemblies...different enzymes and cycling conditions (see Table 1).   Enzyme Temperature Incubation time/cycling...cycling Overhang length T4 DNA ligase 25 °C 1 hour 3-base or 4-base T4 DNA ligase 25 °C ...hours 3-base or 4-base T4 DNA ligase 37 °C 1 hour 3-base or 4-base T4 DNA ligase 37 °C ...Ligase MM 37-16 °C cycling 3-base Table 1: Enzyme and cycling conditions available in DAD tools...rules of thumb for designing your overhang sets. But are those rules the best way to design GGA overhang sets...

  44. Plasmids 101: Control Plasmids

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    Blog Post
    ... that includes the aforementioned internal control and is consistent with the result in Figure 1 could...Gene X using shRNA expressed from a plasmid   Figure 1: Expression Level of Gene X The results shown above...Referring back to the experiment associated with Figure 1, the independent variable is the shRNA. Therefore ...the protein.   In analysis of the result in Figure 1, an internal control, such as the GFP-expressing Plasmid...plasmid.  Let us now revisit our experiment. In Figure 1, it appeared as though the shRNA did not knock down...scientific inquiry and investigation. References: 1. The problem of pseudoreplication in neuroscientific...and should be designed accordingly. If you are trying activate a gene, you should design a control that...

  45. FastCloning: A Newer, Simpler Cloning Technique

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    Blog Post
    ...to the most colonies after transformation. When a 1:1 vector:insert ratio is used in the DpnI digestion... get tips for applying it in your own lab. Table 1: Comparison Between Standard Restriction Cloning and...Connecticut interested in protein science.     References: 1. Celie, Patrick HN, Annabel HA Parret, and Anastassis...PCR cloning method." BMC biotechnology 11.1 (2011): 1. PubMed PMID: 21992524. PubMed Central PMCID: PMC3207894.... Traditional restriction cloning is one of the oldest of these techniques and is a multi-step process...

  46. Lentiviral Vector Guide

    Type
    Guide
    ...typically ranges from 8–10 kb, encoded on RNA (Figure 1). This RNA is reverse transcribed into the provirus...been removed or mutated for increased safety. Figure 1: Wild-type lentivirus genome. Created with BioRender.com...genes (for example, vif , vpr , vpu , and nef for HIV-1). The LTRs flank all of these genes, and anything ...transgene and LTRs (chimeric 5’ LTR) Packaging plasmid 1 — contains gag and pol Packaging plasmid 2 — contains...and third-generation systems, please refer to Table 1. Figure 4: Third-generation lentiviral plasmids. Created...heterologous enhancer/promoter such as CMV or RSV Table 1. Summary of key differences between second- and third-generation...More Lentiviral Vector topics Addgene Protocols pLKO.1 - TRC Cloning Vector Protocols Lentiviral Vector Production...

  47. CRISPR Antimicrobials

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    Blog Post
    ..., they used 1-2 CRISPR crRNAs targeting sequences found only in certain bacteria (Figure 1). Citorik et...from 50% to 11% in just 24 hours.   Figure 1: Single-phage strategies for bacterial CRISPR targeting...genome, and it carries CRISPR machinery targeting 1) the given resistance gene and 2) a second lytic phage...the fight against bacterial superbugs. References 1. Citorik, Robert J., Mimee, Mark, and Timothy K. Lu...Targeted Bacterial Population Editing.” Cell Syst. 1(3):187-196. (2015). PubMed: PMID 26973885. Additional...beta-lactam resistant population to the antibiotic, despite this resistance gene being encoded by a high-copy...currently undergoing a renaissance. Yosef et al. designed a two-phage CRISPR system to resensitize antibiotic-resistant...

  48. Quick Guide to All Things Lentivirus

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    Blog Post
    ...family including human immunodeficiency virus type-1 (HIV-1), can infect both dividing and non-dividing cells...internal promoter, and the desired gene (transfer vector plasmid), 2) the HIV-1 genome containing all viral...to test several protocols that have been described (protocol 1, protocol 2, protocol 3) to find the one...plasmids.  References 1. Miller, A. DUSTY, and C. A. R. O. L. Buttimore. "Redesign of retrovirus packaging...have been devoted to develop efficient and safe HIV-1-derived lentiviral vectors due to their ability to...lentiviral vectors consisted of three plasmids encoding 1) the lentiviral vector genome which was composed of...generation because this vector requires only three HIV-1 genes (gag, pol, and rev) for production. Third generation...

  49. 5 Uncommon Ways to Gain Career Skills During an Uncommon Time

    Type
    Blog Post
    ...need a job. Start now. Informational interviews are 1:1 discussions with someone who has interesting experiences...conversation builds a relationship for the future and the 1:1 format is good for everyone including those that ...out-of-the-box ideas that might jump start your learning. 1. Create a peer mentoring group We don’t all get the...harder to build relationships when you are more desperate or needy so you can’t wait until you imminently...

  50. Plasmids 101: TOPO Cloning

    Type
    Blog Post
    ...the steps needed for TOPO cloning: 1. Create Your PCR Product: Design standard primers (no need to add ... strand and a tyrosyl residue of topoisomerase I (1). If a 5′ hydroxyl group from a different DNA strand...mind that Taq polymerase has an error rate of about 1 in 3,500 bases. Typically polymerases with proofreading...enzyme and Taq, with Taq used in an excess ratio of 10:1. Gel purify your PCR product and incubate it with ...you to see colonies within 8 hours. References: 1. Shuman S. "Recombination mediated by vaccinia virus...
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