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  1. A Look at Addgene's QC Process

    Type
    Blog Post
    ...following sequencing and assembly. In cases where the QC team suspects an assembly issue, they manually revisit..., de novo assembly in Geneious can still provide additional insights to resolve the assembly and determine... Illumina MiSeq. The raw sequencing reads are assembled into a circularized plasmid sequence de novo (...bioinformatics pipeline developed at Addgene. The assembled plasmid sequence is then compared to the information...confirm:  The size of the plasmid to ensure our assembly result is accurate The gene/insert or any major...additional considerations to resolve. Sequencing and assembly issues Sequence discrepancies are not always a... can arise due to errors in the sequencing and assembly pipeline. There are regions of plasmids that are...

  2. Viral Production

    Type
    Collection
    ...filtration through a 0.45 µm polyethersulfone (PES) membrane. The preparations are then aliquoted, frozen, ...

  3. Sonic Hedgehog? Sleeping Beauty? Learn About the Genes Behind Addgene’s Conference Room Names

    Type
    Blog Post
    ...on the surface, giving the embryo a “spiny and prickly look” that resembled a hedgehog (Nusslein-Volhard...eyes. Scientist thought these thick “eyebrows” resembled those of the comedian Groucho Marx, so the gene...Institute! Sonic Hedgehog, an important regulator of embryonic development The largest conference room at Addgene...hedgehog exhibit abnormal segmentation during embryogenesis. Typically, regularly spaced denticles (bristles...bristles) outline the segments of the Drosophila embryo. In flies with non-functional hedgehog, the denticles...gradient exerting a different effect on the developing embryo depending on its concentration. Genetic mutations...mutant fly ovaries had “squiggly” actin fibers that resemble spaghetti. We have two plasmids in our inventory...

  4. Making CRISPR Plasmids Using Fragmid

    Type
    Blog Post
    ... option: Fragmid. Fragmid is a modular vector assembly process designed for a quick, plug-and-play approach...depositor John Doench reports even the greenest members of his lab, high school interns, were able to use...five days. Did you say… easier? Fragmid’s modular assembly structure takes a lot of the design work out of...  Once you have the components you want, simply assemble them into a destination vector via Golden Gate...Whatever individual components you choose will assemble in the expected module order. (It’s also why you...the Fragmid system by sequencing 60 individual assemblies created over five months. When they tested those...were perfectly correct. Because they created the assemblies in duplicate, they were able to confirm that ...

  5. How-to: 5 Steps to a Great Panel Discussion

    Type
    Blog Post
    ...uninteresting, the panel members all say the same thing and one panel member dominates the discussion?... no time for audience questions and an audience member asks a 5 minute question that is really a speech... a fun discussion. People are much better at remembering these things on a relaxed phone call than when...moving and to prevent any one panelist or audience member from taking over. My own personal rule is that ... get things started. Give a couple of audience members a chance to participate. For example, at a recent...nonprofit science organizations, I asked audience members to stand and describe unique nonprofit models that...in the audience. I always try to meet audience members before a panel discussion and often incorporate...

  6. Plasmids 101: Modular Cloning Applications and Kits

    Type
    Blog Post
    ...Modular cloning is a popular DNA assembly tool used to build single- and multi-gene constructs for a ...is. MoClo background MoClo is a clever way to assemble several DNA parts in a specific orientation using...are reusable, saving time and money on future assemblies. If you are new to MoClo, give our MoClo 101 ...Kit has a wide array of DNA parts that can be assembled into individual TUs or very large (up to 20!) ...CIDAR offers a one-pot reaction wherein multi-part assembly can occur. It is quite extensive with a supplemental... most intuitive CRISPR MoClo application is to assemble multiple unique sgRNAs into a multi-TU targeting...save time and money on studies that require many assemblies or that compare the effects of different DNA ...

  7. Antibodies 101: Beyond Surface Labeling

    Type
    Blog Post
    ...diffuse through the cell membrane freely, while others require a permeabilized membrane to enter cells. Depending...Depending on the application, they can be membrane-permeant or membrane-impermeant, and the cells of interest...viability dyes are membrane-impermeant and can only enter cells with compromised plasma membranes — i.e., dead... The targeted epitopes can be motifs within transmembrane proteins, such as receptors, or post-translational... marker of interest is not located on the cell membrane but inside the cell. Due to their chemical nature...nature, antibodies cannot penetrate the cell membrane; hence, intracellular labeling with antibodies requires...Permeabilization, as the name suggests, perforates the cell membrane to allow for antibodies to enter the cell (Figure...

  8. Generating Mouse Models Using CRISPR/Cas9

    Type
    Blog Post
    ... would be injected into developing mouse embryos, the embryos allowed to develop into chimeric mice (with...would first introduce a mutation into a mouse embryonic stem cell (ESC) line, enrich and select for cells...fascinated by CRISPR and rushed to test it. I still remember the day, March 16, 2014, that I got my first preliminary...Streptococcus aureus, is as effective at editing mouse embryos as Cas9 from S. pyogenes and has the advantage ...of fertilized mouse eggs. Or, to avoid handling embryos ex vivo, the components can be electroporated into...Doench’s gRNA design blog post. Finally, always remember that you are working with RNAs, which are prone...payload! Last but not least, when using CRISPR, remember to appreciate the fact that it was first discovered...

  9. Viral Vectors 101: Voltage Indicators

    Type
    Blog Post
    .... They can be targeted to the plasma membrane to measure membrane potential and can also be directed to... the membrane), the voltage sensing component (i.e., rhodopsin naturally resides in the membrane), or ... want to monitor the voltage change across the membrane of cardiac cells. Similarly, neuronal signaling...sensors contained a FP that localized to the plasma membrane due to the addition of several targeting domains...compound that distributes itself within the plasma membrane as well. Another flavor of these tools is Voltron...more lipophilic, increasing its affinity for the membrane, where it will then localize and emit signal from...generate without disturbing the integrity of the membrane, a concern with several of the previously described...

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

    Type
    Blog Post
    ...contacts that bring apposing membranes together and can wrap the membranes into stacked structures including...dimerization.   1. Transmembrane fluorescent protein fusions Transmembrane proteins, i.e. receptors...they cannot tumble in 720°, i.e. they are on a membrane or in an organelle, then their effective concentration...dimerizing fluorescent proteins is illustrated. 1) Membrane proteins fused to FPs that dimerize can form dynamic...integrated into lipid bilayers. Fusion of an FP to a transmembrane protein significantly increases the effective...other. The consequences can be dramatic. For a membrane POI localized to the endoplasmic reticulum, the...tubules can be grossly distorted into dense stacked membranes termed Organized Smooth Endoplasmic Reticulum ...

  11. Cultivating Community Science at BosLab

    Type
    Blog Post
    ...twenty members and volunteers. Transforming citizens into scientists Anyone can become a member of BosLab... if we were teaching classes at the lab, so in December 2015 we held our first intro to bacterial transformation...previous laboratory experience is not required. Our members are skilled with audiovisual media, software engineering...instructor for our intro to biohacking courses. Our members pursue a diverse range of projects at BosLab. Some...experience targeting RNA with small molecules. Other members’ projects range from hacking probiotics to filming.... Community projects at BosLab Not all of our members start out with personal projects in mind. Thus,...encourage more involvement and provide all our members the opportunity to do molecular biology, we’ve ...

  12. CRISPR Guide

    Type
    Collection
    ...against these foreign nucleic acids. Acr family members inhibit CRISPR by a variety of mechanisms, including...Mediated Genetic Screening in Mice with Haploid Embryonic Stem Cells Carrying a Guide RNA Library. Cell ...

  13. REPLACR Mutagenesis: Replacing In Vitro Recombination Methods

    Type
    Blog Post
    ...appropriate vector. Gibson Assembly, GeneArt Cloning, and SLIC can all be used to assemble multiple fragments,...insertions require complicated, often costly in vitro assembly methods. A new method, REPLACR-mutagenesis, harnesses...substitutions - at the same efficiency as Gibson Assembly and GeneArt cloning - but at a much lower cost...mutagenesis methods Current SDM methods rely on in vitro assembly. To make a point mutation, you design two oligonucleotide...al. wondered if, instead of relying on in vitro assembly, they could make the E. coli do the work by using...expensive than gBlock synthesis, and multifragment assembly methods are preferred. No such restriction applies...REPLACR-mutagenesis goes head-to-head with Gibson assembly and GeneArt cloning, the results are favorable...

  14. Gaining Leadership Skills Volunteering at a Professional Organization

    Type
    Blog Post
    ...laboratory work). Committee members have to work professionally with the other members on the team and keep ... according to the needs of my committee members. Some members would need much more guidance, direction...meeting. I first joined a committee as a volunteer member. The NPA runs its committees through Chairs and...and Vice-Chairs that lead them. As a committee member, I volunteered for various tasks as things came up...Directors and liaise with the Executive Director. As members changed, I had to learn how to develop new working...particular style and requirements of the new Board Members. In some instances I felt like I was going back... speaks in “one voice” whilst among individual members, many opinions may exist. I learnt to try and bring...

  15. CRISPR Plasmids - Tagging

    Type
    Collection
    ... use in SapTrap reactions for targeting vector assembly, and the remaining 5 plasmids include Cre and ...

  16. Mouse Modeling, Part 1: Genetically Engineered Mice

    Type
    Blog Post
    ...successfully modified zygotes or embryonic stem cells. Modified embryonic stem cells are injected into a...techniques used to create engineered mouse models at embryonic stages, and the pros and cons of different genome...containing the gene of interest into a zygote, or into embryonic stem cells that are then injected into a blastocyst...insert the gene of interest into a zygote or into embryonic stem cells. Non-homologous recombination incorporates...recombination. A targeting vector is electroporated into embryonic stem cells and this will insert the gene at a ...mice, knockout mice are engineered during the embryonic stem cell stage using one of several techniques...technique is the injection of a targeting vector into embryonic stem cells, which are then injected into a blastocyst...

  17. Networking and Inspiring Young Scientists at Freshers Fairs

    Type
    Blog Post
    ...has 1,283 members, who comprise undergraduates, graduate students, postdocs, faculty members, and the ...rides and other fun activities and even invite members of the public to attend. We thought we’d take some...study chemistry. The society currently has 352 members, ranging from undergrad all the way up to professors... University of Oxford currently has around 700 members and hosts a range of scientific meetings for students...also hosts high level academic debates for its members to become involved with. The society provides talks...in this emerging field. It has gained over 170 members in its first year. The society facilitates opportunities...education and networking opportunities for society members as well as arranging collaborations and conferences...

  18. Plasmids 101: Gram Negative and Positive Bacteria

    Type
    Blog Post
    ...diderms, have a thinner peptidoglycan membrane and an outer lipid membrane. Common gram negative bacteria include...within their membrane, while the stain is washed from the gram negative bacteria’s thinner membrane. Procedure...destroys the outer lipid membrane, and the thin inner peptidoglycan membrane is unable to retain the stain...or monoderms, have a single thick peptidoglycan membrane. Common gram positive bacteria include staphylococci...interaction of CV+ and I- in the thick multilayered membrane of the gram positive bacteria. In a gram negative...

  19. The Crispr Babies Saga Shows the Need for Action, Not More Delays

    Type
    Blog Post
    ...edit a human embryo, or have a woman carry an edited embryo to term. (We should remember that the woman...these embryos is actively being experimented on, too.) However, as research published on December 13 shows..., founding bioethicist of Rogue Bioethics. On November 25, the MIT Technology Review dropped a bombshell...techniques that are sufficiently safe to manipulate an embryo—even if it’s not necessarily via CRISPR/Cas9. This...unethically experiment on humans, and they are not remembered kindly. However, despite many headlines questioning...

  20. Plasmid Tools for Microbiome Studies

    Type
    Blog Post
    ...consists of modular parts that are assembled with Golden Gate Assembly. They built this toolkit upon the...vast, the ability to genetically alter microbiome members is limited and many microbes are difficult to culture...method for genetically altering gut microbiome members in their native habitat (Ronda et al., 2019). They...microbiome systems. Find the toolkit for modular assembly of broad host range plasmids Counting cell division...distributed cell division counting (DCDC), relies on the assembly of fluorescent proteins into one bright particle...fused the red fluorescent protein (RFP) to a self-assembling protein (SAP) such as a bacteriophage shell protein...microbiome bacteria with a toolkit for modular assembly of broad-host-range plasmids." ACS synthetic biology...

  21. Plasmids 101: 5 factors to help you choose the right cloning method

    Type
    Blog Post
    ...allowing you to easily create customized overhangs to assemble up to 6 different fragments in a prescribed order...few dollars per reaction. By comparison, Gibson assembly can cost over $15.00 per reaction if you purchase...purchase a pre-made mix. Nonetheless, Gibson assembly is a highly versatile and efficient way to generate...such as cDNA, PCR fragments, or genomic DNA. Also remember that commercially available cloning kits can often...feature biases the reaction in favor of the desired assembly to make the process close to 100% efficient. Gateway...declines as the number of fragments you’re hoping to assemble increases. Multiple inserts: There are some cloning...through sequence homology. Similarly, Golden Gate assembly allows one to simultaneously complete an ordered...

  22. Congratulations, Deck The Lab winners!

    Type
    Blog Post
    ...their lab’s decoration competition, and each lab member used an impressive variety of lab materials to ...drNBUdlQco — The Tarling-Vallim Lab (@TarlingVallim) December 19, 2020 In third place, the BTC Institute decorated...pic.twitter.com/OOrwo9feyJ — BTC Institute (@BTCInstitute1) December 23, 2020   Deck the Lab: Home Edition Winners...safe and festive Holiday season. 🙌 🥂 🎄 🎉 😷 Remember wear a mask,↔️social distance, 💓show gratitude...pic.twitter.com/ofLI4eD8Zs — Microbigals (@MicrobiGals) December 23, 2020   In second place, Dr. Anne Madden created.../GsAlD6sjM6 — Dr. Anne A Madden (@AnneAMadden) December 23, 2020   In third place is Matthijs Dorst’s...pic.twitter.com/qDylF8TdHH — Matthijs Dorst (@MattNeuro) December 4, 2020 Congratulations to the winners! We’ll...

  23. Hot Plasmids: Fall 2024

    Type
    Blog Post
    ...rate limits its use in systems like the C. elegans embryo, where developmental processes happen within a ...expression levels comparable to GFP during early embryogenesis (Figure 3). mScarlet-I3 also offered clearer...fluorophore-tagged GOLG-4 through eight stages of embryonic development. Representative images of single focal... focal planes through the middle of each embryo are shown, comparing GFP, wrmScarlet (mScarlet), and the...the 11-amino acid BiT fragment of luciferase is embedded in the I10 domain of the human titin protein and...LgBiT luciferase fragment (expressed separately) assemble and catalyze the luminescence reaction (Figure...of luciferase binds to the BiT Tag (also cyan) embedded in the Titin I10 protein (purple) only when Titin...

  24. Management for Scientists: Seeking Feedback

    Type
    Blog Post
    ...excellent, effective communication between all members of a team. As described here in a 2012 Intuit blog...conducts exceptionally well-managed meetings. Members face one another, and their conversations and gestures...well as give, information to make improvements. Members connect directly with one another – not just with...be covered in a future post in this series). Members carry on back-channel or side conversations within...everyone knows what is going on and can contribute. Members periodically break, go exploring outside the team...that brings feedback back to the manager. Team members rarely rush to give honest feedback to the manager...over coffee.  Have regular 1:1 meetings with team members (don’t become known for cancelling these); ask ...

  25. Advice on Career Paths and the Green Card Process for International Researchers and Entrepreneurs

    Type
    Blog Post
    ...achievement Evidence of membership in associations that require their members to demonstrate outstanding...with US immigration in its current state. As of September 2017, USCIS received 116,224 employment based ... position. If you cannot get an Industry H-1B, remember that industry career paths are not the only place...the lab or has won, awards, media reports, and membership in groups related to the foreign national’s field... national award for excellence in your field. Membership in a professional organization – this is not ...assist your PI in the peer review process, but remember you cannot use it as documentary evidence for ...point of view of experts in your field. Please remember, this is not a job reference letter. This letter...

  27. Rett Syndrome

    Type
    Collection
    ...the RSRT along with individual laboratories to assemble a Rett Syndrome plasmid resource for the scientific...

  28. From Lab Bench to Leadership: Transitioning into Management as a PhD

    Type
    Blog Post
    ...requires deliberate preparation and a willingness to embrace new responsibilities. After my second postdoc,...differencesManaging a team isn’t “one size fits all”. Each team member has unique motivations, strengths, and learning...scientist, I worked with a diverse team where some members thrived on independence, while others required ... stay on track and feel supported. Another team member was highly self-motivated and excelled when given...to the communication style of a new, junior team member who had values, working styles, and skills that...discovery project, I noticed tension between two team members with differing approaches—one detail-focused and...permission to explore, reflect on your priorities, and embrace these transitions as an integral part of your broader...

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

    Type
    Blog Post
    ... with SiR-CA (magenta) and outer mitochondrial membrane labeled with SNAP-OMP25 with Atto590-BG (green... 3: Single-molecule tracking of actin filament assembly-regulating proteins in vitro reveals complex events... probes are not very good at crossing the cell membrane. So, you should investigate your options and weigh...self-labeling protein tags are increasingly popular. Embrace the flexibility and switch things up for your next...Biology, 26(4), 584-592.e6. https://doi.org/10.1016/j.chembiol.2019.01.003 Gallagher, S. S., Sable, J. E., Sheetz...Biology, 15(2), 128–136. https://doi.org/10.1016/j.chembiol.2008.01.007 Griffin, B. A., Adams, S. R., & Tsien...https://doi.org/10.1038/nbt765 Lavis, L. (2016, September 8). Better Dyeing Through Chemistry & Small Molecule...

  30. Troubleshooting Your Plasmid Cloning Experiment

    Type
    Blog Post
    ...Gibson Assembly, Golden Gate etc.), and try to find the simplest way to do it (i.e. avoid assembling too ...gel or by nanodrop to assemble the  fragments based on their molar ratio. Assembly of the fragments There...can generate the parts of DNA that you want to assemble together in a combination of different ways according...have a successful and ultimately faster cloning. Remember to quantify the DNA concentration of all the samples...There are multiple ways you can assemble the different parts of a plasmid based on the cloning strategy...

  31. It’s Bioelectric! An Exciting Interdisciplinary Field of Research

    Type
    Blog Post
    ... Pai, Vaibhav P., et al. "Transmembrane voltage potential controls embryonic eye patterning in Xenopus...slightly slower scale.  All living cells with a membrane possess an electrical state. This state, known...is maintained by ion channels and pumps in the membrane. An emerging interdisciplinary field of biology... depolarized (positive) the cell is, the more embryonic, stem cell-like, or cancerous it is (Levin et ...engineering. Planarian worms cut into three segments “remember” which side should grow a head and a tail, and...features increased brightness and improved plasma membrane specificity. Stable Step Function Opsin - A stabilized...

  32. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    ...experiments: Retrieve Ensembl Transcript ID(s) of your favourite gene(s) from Ensembl genome browser: https...https://www.ensembl.org/index.html Design crRNA/ssODN: Using Ensembl Transcript ID(s) as an input query...reagents, we scaled up these tagging experiments by assembling csRNPs in a 96-well plate format and tested parallel...reagents for the knock-in experiment were pre-assembled in a 96-well plate. Glioma stem cells were transfected...be found at Desktop Genetics .For ssODN design, remember to incorporate PAM-blocking mutations (e.g. convert...in bulk and stored in small aliquots at -80 oC. Assemble csRNP: Anneal crRNA and tracrRNA using a PCR block...

  33. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    ...permeabilize the cell membrane to allow antibodies to pass through the cell membrane. Formaldehyde fixation...degree of internal complexity such as extensive membranes produces a greater SSC.  Protein expression, measured...targets, measured by fixing and permeabilizing membranes For intracellular protein targets, users must ... dissolving lipids including those of the cell membrane. In some cases, this can mask epitopes such that...propidium iodide, can only enter cells with disrupted membranes as is the case with dead or dying cells. DNA binding...permeabilization as these methods require damaging the cell membrane and will cause all cells to stain positive. If... amine reactive dyes can only pass through the membrane of dead cells. Once in the cells, amine reactive...

  34. Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene

    Type
    Blog Post
    ...NGS assembles a larger sequence from many smaller sequences, regions with repeats may not assemble correctly...circular assembly. Most of the time, these plasmids are returned as one or two partial assemblies. If we...complete. After the run completes, we begin our assembly process. Again, thanks to our partners at seqWell...from each sample in our pool and individually assembles the reads into a single FASTA sequence that our...that are particularly difficult to sequence and assemble, including GC rich regions. For example, the CAG...

  35. Adapting Toehold Switches to Detect Zika Virus

    Type
    Blog Post
    ...toehold switches for Zika detection We and other members of the lab had been working on advancing the paper-based...Aedes mosquito (Messina et al, 2016). We quickly assembled a team of students, postdocs and professors to...also were careful to set up clear tasks for each member of the team to accomplish so that many elements...Sensor Constructs at Addgene As we report, we have embedded this next generation of toehold switch-based RNA...further develop our rapid prototyping and sensor assembly pipeline for future emerging pathogens or health...synthetic biology, nanomaterials science, and self-assembly for applications in health, energy, and sustainability...Listen to Our Interview with Previous Collin's Lab Member and Sample 6 Cofounder Michael Koeris Learn How...

  36. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ...and plasma membrane reporters as well as secretory proteins or proteins targeted to membrane sub-domains...cleavage upon co-expression and spontaneous self-assembly. In an effort to obtain even more precise temporal...individual reporter constructs or as the fully assembled pMemoryArray.   Yang et al., Nat Methods 2014... Chem Biol. 2012 Apr 20;19(4):507-17. 10.1016/j.chembiol.2012.02.006. 2 Guntas et al., Proc Natl Acad...arrival in their target compartment (e.g. the plasma membrane, the extracellular space, or the lysosomes) can...plasmids with MXS-chaining. Analogous to modular assembly methods, such as BioBricks, the MXS-chaining method... organelles, a cell cycle indicator for mouse embryonic stem cells and a series of inducible promoter ...

  37. Viral Vectors 101: The Retroviral Lifecycle

    Type
    Blog Post
    ...precursor protein. The EMBO Journal, 16(16), 5113–5122. https://doi.org/10.1093/EMBOJ/16.16.5113. PubMed ...commonly called retrovirus) family — of which HIV is a member — may seem like an unlikely candidate to use as...Figure 1 to compare the genomes of these two family members.   Figure 1: Gamma-retrovirus genome (left... RT-deficient genome. In both cases, the lipid membrane is the only component that is not dependent on...genes. It is derived from the host/producer cell membrane, and acquired as an envelope during exocytotic...E., & Teich, N. (1971). Noninfectious ARK mouse embryo cell lines in which each cell has the capacity ...

  38. Plan Your Experiment

    Type
    Collection
    ...off-target effects Can be used to generate transgenic embryos Cas9-gRNA ribonucleoprotein (RNP) complexes Purified...

  39. Caltech Systemic Capsids

    Type
    Collection
    ... pAAV-S5E2-GFP-fGFP E2 regulatory element GFP, membrane-targeted GFP Control Dimidschstein 192552 pAAV-CAG...

  40. Water Bath Protocol

    Type
    Protocol
    ... a water bath with individual slots for tubes, remember that when you add the tube to the slots, it can...
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