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

    Type
    Blog Post
    ...Updated Jun 1, 2021 by Meghan Rego. Reproducible data are key to science, so scientists are used to repeating...the DNA packaged inside the viral particles (Figure 1). Briefly, packaged DNA is extracted from purified.... When contamination is suspected, we proceed to de novo assembly of the unmapped reads, employing our...transduced cells whenever possible. This post will describe our workflow for viral genome sequencing (VGS)...

  2. Plasmids 101: Modular Cloning Applications and Kits

    Type
    Blog Post
    ...Combinatorial Design for Synthetic and Traditional Biology.” ACS Synthetic Biology, vol. 5, no. 1, American...within the mitochondrial matrix.     Figure 1: A chart with MoClo kit recommendations for different..., vol. 4, no. 9, American Chemical Society (ACS), 1 May 2015, pp. 975–86. Crossref, https://doi.org/10.1021...Yeast Mitochondria.” BMC Biotechnology, vol. 17, no. 1, Springer Science and Business Media LLC, 13 Nov. ...Editing in Plants.” BMC Plant Biology, vol. 20, no. 1, Springer Science and Business Media LLC, 23 Apr. ...Arabidopsis Mutants.” The Plant Journal, vol. 106, no. 1, Wiley, 25 Mar. 2021, pp. 8–22. Crossref, https://...Cloning Platform.” Scientific Reports, vol. 9, no. 1, Springer Science and Business Media LLC, 12 July ...

  3. Plasmids 101: Modular Cloning

    Type
    Blog Post
    .... Level 1: Assembly of multiple Level 0 parts into TUs. Level 2: Assembly of multiple Level 1 TUs into...: promoter, gene, and terminator.     Fig 1. MoClo enables individual parts to be assembled into... peptide can easily be tested by generating Level 1 TUs with unique promoters, and the ideal phosphatase...cloning, or MoClo, is a systematic cloning strategy designed by synthetic biologists to perform large scale...specifically refers to the organizational schema described above, and not any particular cloning technique...example of an individual part, this category also includes promoters, UTRs, terminators, etc. Once selected...behave in a cell. For unpredictable cases, MoClo provides two answers: first, sets of parts with characterized...

  4. CRISPR Guide

    Type
    Guide
    ...M. M., Semenova, E., Severinov, K., De Vos, W. M., Dame, R. T., De Vries, R., Brouns, S. J., & Van Der... most popular genome engineering approach. Figure 1: Overview of the basic CRISPR mechanism Engineered...from Staphylococcus aureus ) has a coding sequence ~1 kb shorter than SpCas9 while retaining the same basic...lentiviral transfer vectors (Figure 8B). Libraries come in 1-vector systems, in which Cas9 is included in the gRNA-containing...Cascade ( C RISPR- as sociated c omplex for a ntiviral de fense) complex to initiate DNA degradation. Cascade...Cascade C RISPR- as sociated c omplex for a ntiviral de fense; a complex of multiple Cas enzymes including...efficiency and fidelity. Nature Communications , 13 (1), 1425. PMID: 35301321 Edraki, A., Mir, A., Ibraheim...

  5. Tips from the Repository Trenches: Using Barcodes to Track Samples

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    Blog Post
    ...necessary.   Tips for barcoding and tracking samples 1. Start with the tube - Barcoded tubes come in many...all of these samples have the 2D barcodes mentioned above. These barcodes identify what type of sample is...tubes. 1D barcodes are better for tubes that are meant to be read one at a time. 1D barcodes can be purchased...the tubes’ physical location in the lab. These barcodes additionally allow us to store multiple types ...fresh media with antibiotics at each stage. The barcodes and LIMS help us track each sample, but we also... a petri dish plate that is also barcoded. The barcodes on both of these are scanned into our LIMS system...samples within your lab, we highly recommend using barcodes! Barcoded tubes are easily accessible and can ...

  6. Viral Vectors 101: Parts of the AAV Packaging Plasmid

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    Blog Post
    ... The three VP proteins assemble into capsid at a 1:1:10 ratio (VP1:VP2:VP3). There are eleven naturally...closer look at the rep and cap genes. Figure 1. The RepCap plasmid and its products. Created with...plasmid The packaging plasmid aka the RepCap plasmid encodes two genes: the replication (rep) gene and the capsid....com   Replication (rep) gene The rep gene encodes four proteins that are required for viral genome...cross-packaging.  Capsid (cap) gene The cap gene encodes three AAV viral capsid proteins VP1, VP2, and VP3...type tropism. Introducing changes and/or short peptides sequences into these variable loops is one method...method for changing a capsid’s tropism.  cap also encodes the Assembly-Activating Protein (AAP) and the recently...

  7. Cancer and the Immune System: Deciphering the Relationship

    Type
    Blog Post
    ...such as IL-8, IFNγ, and colony stimulating factor 1 (CSF-1). Additionally, tumor cells recruit regulatory...introduced the “Three Es of Immunoediting” nomenclature. 1. Eliminate - Latent tumors are recognized by effector...the tumor, activates hypoxia inducing factor (HIF-1), which promotes the production of VEGF to increase...This study used CRISPR gene editing to knockout PD-1 on immune cells harvested from patients and thereby...exploring new avenues in healthcare.   References 1. Vinay, Dass S., et al. "Immune evasion in cancer:...lymphoblastic leukaemia in children and young adults: a phase 1 dose-escalation trial." The Lancet 385.9967 (2015)... recognition are a few methods by which tumors sidestep immune surveillance: Suppressing Function Antigen...

  8. Plasmids 101: Broad Host Range Plasmids

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    Blog Post
    ...Table 1 has a detailed list of the bacterial species these plasmids are compatible with.  Table 1: Non-...reduced metabolic load (Meyer 2009). Figure 1: Idealized examples of a narrow host range plasmid...for DNA cloning in Gram-negative bacteria. Gene, 70(1), 191–197. https://doi.org/10.1016/0378-1119(88)90117... N.J.), 765, 327–343. https://doi.org/10.1007/978-1-61779-197-0_19  Meyer, R. (2009). Replication and ... meliloti, Rhodobacter sphaeroides, R. capsulatus, Rhodopseudomonas spheroides, Salmonella spp., Serratia...Brevibacterium methylicum, Caulobacter crescentus, Desulfovibrio vulgaris, Erwinia carotovora, E. chrysanthemi...meliloti, R. leguminosarum by. viciae, Rhodobacter sphaeroides, Salmonella typhimurium, Vibrio cholerae, Xanthomonas...

  9. Three Key Considerations for Precise Neuronal Targeting Using AAV Technologies

    Type
    Blog Post
    ...main takeaways from the paper for designing experiments with AAV: 1. Getting your viral vector into your... administration routes for neuroscience see Table 1 from the paper. 2. Manipulate your cell of interest...information from a region. In the example shown in Figure 1, injected retrograde AAVs at one location result in...AAV from our viral service can share experimental design and data. With these different approaches to open...

  10. Finding and Joining Your Dream Lab

    Type
    Blog Post
    ...choosing direction of a project. If you expect more 1:1 time with the advisor, you should choose accordingly...potential hire visits the lab for a full day, gives a 1 hour talk about his or her interests, and spends one-on-one...that supports both paths – you won't know in year 1 what you will want in year 6. Have lab alumni taken... a Good Mentor for Scientists". This post also includes a useful list of questions for a candidate to ...

  11. Plasmids 101: Knockout/Knock-In Plasmids

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    Blog Post
    ... the example shown in Figure 1, large sequences corresponding to exons 1 and 3 of the targeted gene have...through an experiment designed to knock out exon 2 of a given gene.   Figure 1: A knockout targeting... the Neomycin resistance (NeoR) gene between exon 1 and 3 of the targeted gene. Upon recombination, the...often use the Cre/Lox system described earlier. In this case, you design your targeting vector such that...replacing it or disrupting it with an piece of DNA designed in the lab. Specially constructed plasmids can...template with the endogenous gene. Here, we'll describe the techniques and the plasmids used to inactivate...inactivate specific genes in mammalian cells. Despite the popularity of CRISPR-based knockout/knock-in systems...

  12. New Acoustic Reporter Genes: Ultrasound Imaging of Gene Expression

    Type
    Blog Post
    ...under a wide range of inducer concentrations (0.1-1% L-arabinose) without signs of toxicity or burden ...bacteria have colonized (Fig. 1c).     Fig. 1: Expressing bARGSer in E. coli. (a) Plasmid diagram...ultrasound contrast. In all the cell lines we tested, 1 µg/mL doxycycline was sufficient to saturate expression...engineering a cell line, cotransfect the mARG cassettes 2:1 (A:N-V) with PiggyBac transposase and outgrow the ... tumors and tumor-homing bacteria. Nat Biotechnol 1–13 (2023) doi:10.1038/s41587-022-01581-y  ...to distinguish from background tissue unless destructive ultrasound imaging was used. We set out to develop...acoustic properties of these gas vesicles made non-destructive specific imaging in tissue difficult. The first-generation...

  13. Plasmids 101: Using Transposons in the Lab

    Type
    Blog Post
    ...Transposons: The jumping genes. Nature Education 1(1):204. https://www.nature.com/scitable/topicpage/transposons-the-jumping-genes...poly(A). ORF2 encodes endonuclease and reverse transcriptase activities.    Figure 1: Overview of...cholerae that encodes a Type I-F CRISPR-Cas system. This system has four major components: 1) a CRISPR RNA... They transpose by a ‘copy-paste’ mechanism (Fig. 1). They first copy themselves as RNA transcripts and...their host’s genome.   There are two types of Class 1 TEs: those with long terminal repeats (LTRs) and those... sequence that moves around. This DNA sequence encodes the proteins the transposon needs for transposition...contain two genes: gag and pol. The pol polyprotein encodes the reverse transcriptase and integrase enzymes...

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

    Type
    Blog Post
    ...applications. Figure 1: Schematic of an RNAi-inducing transgene. Typically, the desired target of RNAi is...dsRNA via an RNase III enzyme named Dicer (Figure 1). The plant kingdom has expanded its repertoire of...in the biogenesis of different small RNAs (Figure 1). These siRNAs can exert their gene silencing effect...modifications of complementary gene sequences (Figure 1). As a result, gene silencing can be easily induced...complementary to your gene target as inverted repeats (Figure 1). With this technique, multiple genes can be simultaneously...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...

  15. Educational Planarian Tools for Teaching Developmental Biology Techniques

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    Blog Post
    ...experiments. A recent publication [1] from the Sánchez lab describes the plasmids and protocols required...available through the deposit) are described in the Sánchez lab publication [1] so that the students performing...can easily investigate in planarians. References 1. Alice Accorsi, Monique M. Williams, Eric J. Ross,...Proceedings of the National Academy of Sciences 100.suppl 1 (2003): 11861-11865. PubMed PMID: 12917490. PubMed...companion resource called “cutting class” which provides more detailed materials and protocol resources...synthetically made RNA (riboprobes) with modified nucleotides (for example, Digoxigenin-labelled Uracil) that... molecular cloning to insert the cDNA into the desired plasmid backbone. The Sánchez lab plasmids allow...

  16. Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins

    Type
    Blog Post
    ...discovered such as Kaede and PS-CFP2 (see Table 1). Table 1: Properties of Selected Irreversible Photoactivatable...preserve cellular structures (0.5-1% osmium tetroxide). These conditions destroy most PA-FPs, but, in 2015, ...) by mutating Threonine 203 to Histidine (T203H) (1). wtGFP normally contains a mixed population of neutral...in fluorescence when illuminated with blue light (1). Similar to PA-GFP, scientists later developed red...with extraordinary molecular detail.   References 1.Patterson, George H., and Jennifer Lippincott-Schwartz...is believing." Annu. Rev. Biomed. Eng. 10 (2008): 1-38. PubMed PMID:18647110. Additional Resources on ...to green). For example, mEosFP (named after the goddess of dawn in Greek mythology) switches from green...

  17. A Needle in a Base-Stack: Cas9 Structural Biology

    Type
    Blog Post
    ...2017). Within an interior cavity, Cas9 grips nucleotides 1-10 of the guide RNA, prearranged for base pairing... basic parts of the Cas9 enzyme.   Figure 1: A cartoon depiction of Cas9’s two major lobes, REC...the CRISPR system (Nishimasu et al., 2014). Table 1. Summary of Cas9 domains, the lobe each domain belongs...state (PDB ID 4CMP) with domains colored as in Figure 1. HNH, red; RuvC, orange; CTD, yellow; REC lobe, light... on itself at the bottom of the image. Stem loops 1 and 2 extend horizontally across both lobes. The guide...outside of the protein, spanning both lobes. Stem loop 1, nestled between the REC and CTD domains, is required...343–347. https://doi.org/10.1038/s41586-022-04470-1 Cofsky, J. C., Soczek, K. M., Knott, G. J., Nogales...

  18. Fluorescent Proteins 101: Introduction to FRET

    Type
    Blog Post
    ...acceptor fluorophore. The typical steps in FRET are: 1. Donor fluorophore excitation by absorption of a photon... overlap, the fluorophores must be located within 1-10nm of each other and be oriented appropriately for... from various papers to make the decision easier. 1. Müller, Sara M., et al. "Quantification of Förster...diseases and even assist in drug discovery. References 1. Baird, Geoffrey S., et al. "Circular permutation ...Resonance Energy Transfer (FRET) was originally described by Theodor Förster in 1948 as a variation of the... observed light emission by fluorescence. The widespread use of FRET with fluorescent molecules, including...FRET probe, try searching PubMed for articles describing the FRET tool that you are looking for and check...

  19. Magnetic Control of Proteins: More than a Dream

    Type
    Blog Post
    ...the sample (by less than 1%) and recovered after the magnet was removed (Figure 1). At first, Andrew and...since the initial report in 2023.)     Figure 1: Fluorescence intensity of EGFP-FlavinTag in E. coli...that could be expressed and purified. Although a ~1% change in signal might not make a very powerful tool...Hayward et al. 2024 under CC-BY license.   Video 1: Fluorescence movie of E. coli expressing MagLOV while...Maria were encouraged by previous reports that described magnetic responses of cryptochrome proteins or...

  20. Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR

    Type
    Blog Post
    ...by PCR or next-generation sequencing. Figure 1: Overview of the key steps of the CAPTURE method. ...ligases Expression system Stable cell line expressing 1) FLAG-biotin-tagged dCas9, 2) BirA, and 3) one or ...or more gRNAs Two separate lentiviruses delivering 1) a BioTAP-tagged dCas9 and an eGFP tag, and 2) gRNAs...(s) Cell lines Cell lines, primary cells Table 1: Key differences between original CAPTURE and CAPTURE...The Xu lab presented three ways to use CAPTURE 2.0: 1. Multiplex capture of chromatin interactions The Xu...Variant for Your Next Experiment Learn More About Designing gRNAs Against Your Favorite CREs Here Additional...

  21. Antibodies 101: Affinity Tags

    Type
    Blog Post
    ...118). Humana Press. https://doi.org/10.1385/1-59259-301-1:99 Resources on Addgene.org Addgene antibodies...antibody detection is desired… and yes, we have an antibody for that!   Fig. 1 - Diagram of His-tagged...You’ve designed the perfect experiment – controls, conditions, and everything in between – now all you...This is where affinity tags come in – they are peptides which do not naturally exist in most expression...protein for downstream applications Below, we describe a few of the most common tags available. Maltose...antibody is an alternative to amylose resin and also provides a visual detection mechanism via applications ...

  22. RUBY-Red Siliques

    Type
    Blog Post
    ...collected from heterozygous individuals. Only 1/4 of the descendants of the heterozygous plants, therefore, ...in all other plants, anthocyanins.   Figure 1: An adult plant expressing RUBY. Photos by Andrew ... (2020). https://doi.org/10.1038/s41438-020-00390-1 Additional resources on the Addgene blog  CRISPR 101... Antibiotic selection would only then kill the desired plants! PCR or fluorescent markers are currently... have similar drawbacks, often requiring the destruction of plants or tissue for GUS staining or are not...from these plants will be green, containing the desired mutation but lacking both RUBY and the construct...compared to other markers lends itself well to nondestructive selection. If RUBY was integrated into a CRISPR...

  23. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    ... emailed submission) in the Deposit menu: Figure 1: Begin the deposit process by clicking "Submit Online...cases Please contact us at [email protected] or +1 (617)-ADDGENE (233-4363) if your deposit falls into...Addgene?" or contact us at [email protected] or +1 (617)-ADDGENE (233-4363) with additional questions...strain. Please contact us at [email protected] or +1 (617)-ADDGENE (233-4363) if you’d like to distribute...page so that any data you enter is not lost.   Step 1: Sequences, Maps, and Files Please provide any full...a 1.5 mL microfuge tube at a concentration of 0.1–1 µg/µL (or bacterial streaks, if you prefer). If you...preprint title or a general description of the deposit. Select your desired distribution status: Hold...

  24. Generating Mouse Models Using CRISPR/Cas9

    Type
    Blog Post
    ...Sullender M, Ebert BL, Xavier RJ, Root DE (2014) Rational design of highly active sgRNAs for CRISPR-Cas9...Last updated on Oct 1, 2020 by Aliyah Weinstein. This post was contributed by guest bloggers, Wenning...process check out our Mouse Modeling blog posts: Part 1: Genetically Engineered Mice and Part 2: Breeding ...LO, Morgan JL, Juan WC, Roca X, Ong ST, Bergstrom DE (2018) Viable Mice with Extensive Gene Humanization... mouse genome is made up of 3 x 109 nucleotides (nt), and encodes 23,000 or so genes. It would be great...guide you in gRNA design. We have been using Benchling, but there are many other gRNA design tools available...for cells that had successfully incorporated the desired mutation, and then derive mice from these engineered...

  25. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

    Type
    Blog Post
    ...occur. While the Brainbow-1 system employs Cre-mediated deletion, the design of Brainbow-2.1 cleverly ...homologous, preventing design of antibodies specific to each fluorophore. Third, Brainbow-1 and Brainbow-2 each...the Brainbow mouse neural labeling system. Brainbow-1 and -2 have been further refined to create Brainbow...dendritic processes not previously visible with Brainbow-1 and -2. The general structure of Brainbow-1.0 is retained...Rheenen J, Simons BD, Clevers H. Cell. 2010 Oct 1;143(1):134-44. PubMed. Drosophila Brainbow: a recombinase-based...R26R-Confetti described in Hugo J. Snippert, et al. (2010) and the MAGIC Marker strategy described in Karine...immediately following the promoter. The STOP cassette includes a mutant YFP that does not fluoresce, but can ...

  26. Antibodies 101: Conventional vs Spectral Flow Cytometry

    Type
    Blog Post
    ...detector for that fluorophore. For example, in figure 1, the FITC signal is spilling over into the primary...spectra to use for compensation.     Figure 1: The emission spectra and filters for FITC and PE ...in the panel. This complexity index ranges from 0-1, with a value of “0” indicating those markers are ...completely distinct from one another, and a value of “1” indicating those markers are identical and completely...positive and negative populations, Careful panel design In both conventional and spectral flow cytometry...indistinguishable. This tool is of particular value when designing a panel.     Figure 3: Similarity index...if markers are co-expressed in your cells when designing your panel. If you have two highly overlapping...

  27. Selecting Your Plasmid Purification Kit

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    Blog Post
    ...amounts of high quality DNA. Figure 1 illustrates the three grades of DNA you may need based on their ... kits are designed to accommodate specific volume ranges of culture as their input (Table 1). When you...miniprep kit for example. These kits are designed to handle ~1–5 mL of liquid culture. Using a higher volume...their relative quality and quantity.   Figure 1: Plasmid preps fall into different categories based...beads and a suitable magnet as you scale up. Table 1: Standard Prep Kit Capacities* Prep size Amount...Amount of culture Approximate DNA yield** Miniprep 1–5 mL 5–50 µg Midiprep 25–100 mL 20–200 µg Maxiprep...components (i.e. buffers, columns, or beads) are designed to work together — no mixing and matching!   ...

  28. Pooled Library Amplifications

    Type
    Blog Post
    .... Table 1 provides an overview of pooled library protocols you can use as a reference. Table 1: Examples... be more difficult to transform.    Figure 1: Factors to consider when creating a pooled library...etc. The DNA amount can be anywhere from 10 ng to 1 μg of DNA. It often correlates with the size of the...(Are the inserts of different sizes?), and the desired coverage for the library (How important is it that...  The most important factor to consider is your desired library coverage, since this will help decide what...should always experimentally test the protocol you design to ensure that it works for your library before...Addgene. If you have any specific questions about designing your library amplification protocol, feel free...

  29. Chromoproteins: Colorful Proteins For Molecular Biology Experiments

    Type
    Blog Post
    ...attract fish for them to prey upon (Figure 1). Figure 1: The sea anemone's red color can be used ...synthetic biology. Journal of Biological Engineering, 12(1). https://doi.org/10.1186/s13036-018-0100-0 Murakoshi...expression in living cells. Scientific Reports, 9(1). https://doi.org/10.1038/s41598-019-48604-4 Salih...fluorescent proteins, it’s important to note that despite their distinction by name, chromoproteins are actually...chromoproteins have a shorter history. In 1987, scientists described an unusual blue-colored protein from the jellyfish... chromoprotein plasmid that combines the most desirable features: intense color, fast maturation, and ...recombination events: The plasmid carrying the desired genetic modification integrates into the genome...

  30. Optogenetics Guide

    Type
    Guide
    ...algae Chlamydomonas reinhardtii . Channelrhodopsin-1 (ChR1) is excited by blue light and permits nonspecific...based on the LOV2 domain of Avena sativa phototropin 1 LOVETRAP reversibly sequester and release proteins...concept of optogenetics. 2012 Prog Brain Res. 196: 1-28. PMID 22341318 Gradinaru V, Zhang F, Ramakrishnan...diversifying and extending optogenetics. Cell. 196:1-28. PMID 20303157 Han X, Boyden ES. 2007 Multiple-...Limitations and Future Developments. Exp Physiol. 96(1): 19–25. PMID 20621963 Mattis J, Tye KM, Ferenczi ... Science Guides Optogenetics Guide Optogenetics Guide Jump To: ...cell membrane. Microbial opsins, such as those described below, can be targeted and expressed in specific...

  31. Mouse Modeling, Part 1: Genetically Engineered Mice

    Type
    Blog Post
    ...reporter gene, which replaces the targeted gene and provides a way to track successfully engineered cells. ...genotype each animal to identify the ones with the desired genotype. Inducible and conditional systems Conditional...

  32. Cpf1 Update: Comparison to Cas9 and NgAgo

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    Blog Post
    ...on-target and off-target editing efficiency Figure 1: Effects of (A) double and (B) single base pair gRNA-target...that Cpf1 can tolerate mismatches at gRNA positions 1, 8, 9, and 19-23. Accordingly, the 3’ end of the gRNA...Most gRNAs directed low-frequency Cpf1 cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave...fits into the genome editing landscape. References 1. Kim D, Kim J, Hur JK, Been KW, Yoon SH, Kim JS. Genome-wide...for Cas9 (42 nt vs 100 nt.) However, the newly described non-CRISPR protein NgAgo is smaller than any of... of these options (2.7 kb with 24 nt guides), so it has the potential to eclipse any of the CRISPR nucleases...to examine Cpf1 off-target editing. First, they designed gRNAs with single and double mismatches throughout...

  33. PCR Applications for Copy Number Variation Assays

    Type
    Blog Post
    ...against the limitations of the assay.      Fig. 1: A schematic representation of Sybr Green chemistry...Methods Mol Biol. 2018;1768:143-160. doi: 10.1007/978-1-4939-7778-9_9. PMID: 29717442. Arya M, Shergill IS...amplification of Plasmodium falciparum multidrug resistance-1 gene (pfmdr1). J Parasitol. 2011 Oct;97(5):939-42....gamma receptor genes FCGR3A and FCGR3B. PloS one, 10(1), e0116791. https://doi.org/10.1371/journal.pone.0116791...results can be affected by DNA quality and primer design, particularly for amplicons under 100 bp (Ma, 2015... concern, look for the lowest-cost option that provides you confidence in the results you need.  Whichever...by running the product on a gel. And only ddPCR provides absolute quantitative data, instead of relative...

  34. Your Lentiviral Plasmid FAQs Answered

    Type
    Blog Post
    ... vectors are: 1) the potential generation of replication competent virus [usually HIV-1]; and 2) the potential...systems have been developed over the years based on HIV-1; however, the 1st-generation vectors are never used...which has been shown to repress expression of HIV-1 LTRs (more information is available here). 2nd vs...those seen when using a plasmid backbone that was designed specifically for transient expression (find some...expression of viral constructs is a useful tool as it provides a means to quickly check that a construct is functional...3rd-generation transfer plasmids have a chimeric 5'LTR that includes a CMV or RSV promoter as well as a portion of ...those three genes plus tat. The envelope plasmid provides a heterologous envelope for pseudotyping and is...

  35. Choosing a Good Mentor for Scientists

    Type
    Blog Post
    ...setting out of the lab (coffee, lunch or beer) and in 1:1 conversations. This creates a level of familiarity... other posts in our Science Mentors series: Part 1: "What Makes a Good Mentor?" and 6 More FAQs About...PDF at the end of this blog post. This document includes a list of questions to ask your potential advisors...some good pre-work you can find a lab that will deserve your hard work and an advisor that will be a lifelong...

  36. Optogenetics + CRISPR, Using Light to Control Genome Editing

    Type
    Blog Post
    ...2 (CRY2) and calcium and integrin-binding protein 1 (CIB1) proteins. The goal of both groups was to create... the Sata lab is composed of two fusion proteins: 1) the genomic anchor - an inactive, dead Cas9 protein...combination. The optimized LACE system consisted of: 1) CIBN-dCas9-CIBN, where CIBN is the N-terminal fragment.... crassa) (Kawano et al., 2015). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and...fold-induction of Cas9 activity (16.4-fold). This paCas9-1 light-inducible system was able to recognize the same...flCas9). When triggered by blue light (470nm), paCas9-1 induced indel mutations via NHEJ (frequency of 20.5%...background activity of the system by modifying paCas9-1 using nMagC714 instead of nMagHigh1-C714, generating...

  37. 9 Tips to Achieve Success in Academia

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    Blog Post
    ...leading professors for their top 3 tips. Tom Ellis 1. Keep up with the literature. He recommends setting...research and with getting published.” Connie Cepko 1. Work hard. There will be periods when opportunities...time comes to work with full force. George Church 1. “Initial and sustained motivation is a huge component...entertainment, politics, K-12, etc.” Addgene's team includes many PhD scientists who have stepped away from...

  38. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    ...by mating with Cre-expressing mice. Figure 1: (A) Schematic of the Cre-dependent SPH construct ... alpha (aCaMKII; excitatory neurons) and Synapsin-1 (pan-neuronal) promoters (Figure 1b), and sgRNAs can...ubiquitous expression of sgRNA(s) and mCherry (vector 1) with neuron-specific expression of dCas9-VPR (Figure...Neuron-specific expression is mediated by the synapsin 1 (SYN) promoter, which was superior in its ability ...Patrick Miller-Rhodes from the University of Rochester Medical Center. Patrick Miller-Rhodes is currently...This post was contributed by Patrick Miller-Rhodes, a Ruth L. Kirschstein NRSA Predoctoral Fellow at ...this technological gap, a trio of recent papers describe the development of CRISPR activation (CRISPRa)...

  39. Rosella: A Fluorescent pH-Biosensor for Studying Autophagy

    Type
    Blog Post
    ...’s fused to the C-terminus of DsRed.T3. See Table 1 for a summary of Rosella’s excitation and emission...fluorescence Rosella emits based on its localization. Table 1: Summary of Rosella’s excitation and emission spectra... variants are available from Addgene. References 1. Rosado, C., Mijaljica, D., Hatzinisiriou, I., Prescott...single fluorescent biosensor. Scientific Reports,5(1). doi:10.1038/srep12397. PubMed PMID: 26215030. PubMed... phosphatase activity: In yeast, the PHO8 gene encodes a vacuolar alkaline phosphatase. Normally PHO8 ...

  40. Viral Vectors 101: Systemic Capsids

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    Blog Post
    ...total dose between 1 x 1011 and 5 x 1011 vg per animal (i.e., 10 and 50 µL of vector at 1 x 1013 vg/mL titer... and 1 x 1012 vg*. Most systemic capsids are easy to produce in high titer, but higher doses (>1 x 1012...selecting a capsid, while Table 1 gives a brief overview of published descriptions of commonly used or emerging...before using it in an application.     Figure 1: AAV toolkit for targeted gene delivery to the brain...Capsids page to learn more about that collection. Table 1: Systemic capsid plasmids available from Addgene ...AAV9. At lower viral titers than AAV-PHP.B (e.g., 1 x 1011 vg/mouse), AAV-PHP.eB can transduce a greater...Diverse Brain Cells. Annual Review of Neuroscience, 45(1), 447–469. https://doi.org/10.1146/annurev-neuro-111020...

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

    Type
    Blog Post
    ... to other technologies like CRISPR. Figure 1: An overview of the general schematic of CRISPR/Cas... can be detected – in the zeptomolar range Time: ~1 hour Equipment required: 37°C water bath and 42°C ...nearly single-molecule sensitive. In the case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification... Not mentioned in the paper, likely approximately 1-2 hours Equipment needed: Mini PCR machines by DIY-Bio...Sensitivity: – 110 femtomolar  Time: Not mentioned, likely ~1-2 hours Equipment needed: PCR machines/Heat blocks...flattening the curve, it has become evident that widespread testing is crucial in controlling this pandemic...one temperature. Two Cas12a-crRNA complexes are designed, each binding to one strand of the reverse-transcribed...

  42. Plasmids 101: Restriction Cloning

    Type
    Blog Post
    ...well, so let’s go over the details step-by-step. 1. Digestion Set up restriction digests for your insert...recipient plasmid : insert ratio” of approximately 1:3. Since the number of base pairs for each varies,...cells. For most standard cloning, you can transform 1-2μl of your ligation reaction into competent cells...at minimum, two transformations after a ligation: 1. Control Transformation containing the ligation mixture...result in few colonies on both plates (unsuccessful 1), in a vector alone plate with many more colonies ...single enzyme if your insert is flanked on both sides by that enzyme’s restriction sites, but the insert...the orientation of your insert. You may want to design a diagnostic digest for this purpose. Ideally, ...

  43. Multifunctional Neural Fibers: Modulation from Gut to Brain

    Type
    Blog Post
    ...Fun fact: A 1 cm block of polymer can generate many meters of fibers!     Fig. 1 - Overview ... and brain neural circuits. Nature Biotechnology, 1–13. https://doi.org/10.1038/s41587-023-01833-5 Resources...blocks of polymers are heated up and pulled to a desired thickness. The fibers are drawn with microelectronics...whistles’ – including microscale light emitting diodes, thermal sensors, and microfluidic channels. These...using NeuroStack and the fibers as previously described, then measured electrophysiological potential ...

  44. AAVs in Retinal Gene Therapy

    Type
    Blog Post
    ...and Therapeutic Purposes. Annu Rev Virol. 2014 Nov;1(1):427-51. DOI: 10.1146/annurev-virology-031413-085355...personalized medicine. References and further reading 1. FDA approves gene therapy for blindness 2. Gene Therapy... Med Biol. 2014; 801: 687–693.  doi:  10.1007/978-1-4614-3209-8_86. PubMed PMID: 24664759. PubMed Central...whole field to a grinding stop and only now, 2 decades later, is it finally back in the limelight. On ...(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 ...

  45. Lentiviral CRISPR Libraries Enable Genome-Scale, Knockout Screening

    Type
    Blog Post
    ...plasmids. The Zhang lab, on the other hand, uses a 1 plasmid, single infection system. Genome wide mouse...X, Scott DA, Mikkelson T, Heckl D, Ebert BL, Root DE, Doench JG, Zhang F. Science. 2013 Dec 12. (PubMed...Wang’s library is designed to target a smaller set of genes and it includes more guides per gene. The Sabatini...Two new human lentiviral CRISPR library systems described in companion papers in Science in December were...Additionally, a concurrent Nature Biotechnology article describes the development of a mouse lentiviral CRISPR library...

  46. Expanded Access to Rabies Plasmids

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    Blog Post
    ... export license applications for VSV and SARS-COV-1 plasmids. Export control license process    An export...charged.  Timelines  We have offered VSV and SARS-COV-1 plasmids under this application process since May ...institution. Requesters will need to provide a brief description of the applicable research project when purchasing...

  47. Bacterial Expression Systems

    Type
    Collection
    ...expression inducible by red light. pCph8 50552 pLtetO-1 Anhydrotetracycline Jeffrey Tabor Anhydrotetracycline... cleavage site. Includes sites for LIC cloning, and a "stuffer" fragment that includes the SacB gene, ... like… Plasmids 101 Addgene's Molecular Biology Guides Plasmids 101: E. coli strains for protein expression...! Plasmid ID Promoter Epitope Tag(s) ID Purpose pDEST-HisMBP 11085 Tac (lactose/IPTG inducible) MBP, 6x...and an MBP tag. Compatible with Gateway cloning. pDEST-periHisMBP 11086 Tac (lactose/IPTG inducible) MBP...molecule, 3OC6HSL, from a promoter repressed by EsaR. Designed to be used with the pAC-EsaR Series . pAC-EsaR...Christopher Voigt When combined with pPLPCB(S), encodes a green light responsive sensor protein that activates...

  48. The AAVantages of AAV in CRISPR Screens

    Type
    Blog Post
    ...NGS) to identify genes of interest. Figure 1: Overview of in vitro AAV CRISPR screens. Created ...certain experiments. AAV can be safely handled at BSL-1, while lentivirus requires a BSL-2 setup. This requires...CRISPR-induced DNA breaks. Nature Communications, 10(1). https://doi.org/10.1038/s41467-019-12449-2 Pupo,...diseases. Signal Transduction and Targeted Therapy, 9(1). https://doi.org/10.1038/s41392-024-01780-w Additional... This vastly increases the possibilities when designing screens. Each serotype has its own tropism, or...experiment. Additional considerations One of the downsides to using AAV is the limited packaging capacity...Adames, A. C., Saur, D., & Rad, R. (2022). Tutorial: design and execution of CRISPR in vivo screens. Nature...

  49. The Materials Science of Optogenetics Experiments

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    Blog Post
    ... and materials required for each [1, 2]. This protocol [3] provides, in exquisite detail, the various ...follow him on Twitter @derekpsimonphd.    References 1. Gutierrez DV, et al. Optogenetic control of motor...manipulation of neural circuits. Nature protocols. 2012;7(1):12-23. doi: 10.1038/nprot.2011.413. PubMed PMID: ...freely moving mice. Neuroscience research. 2011;70(1):124-7. doi: 10.1016/j.neures.2011.01.007. PubMed ...

  50. A Look at Addgene's QC Process

    Type
    Blog Post
    ...If a depositor provides a sequence for the plasmid, QC scientists will first align the de novo NGS result...experiments, to false conclusions.  Figure 1. Potential points where DNA errors can be introduced...are assembled into a circularized plasmid sequence de novo (without the use of a reference sequence) using...sequence, if one was provided. If no reference is given, de novo assembly in Geneious can still provide additional...plasmids in our collection. This standardization provides security for requestors, ensuring that the plasmid...types of data for each of their plasmids, from descriptions of key components of the plasmids to partial...believe may affect the function of the plasmid. This includes point mutations, frame shifts, missing elements...
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