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  1. Early Career Researcher Toolbox: Free Online Molecular Biology Tools

    Type
    Blog Post
    ...Cloning Design primers for Gibson Assembly and NEB HiFi DNA Assembly  OligoAnalyzer Tool from IDT: For... vector database: Find sequences for plasmids assembled from publications and commercially available sources...other primer characteristics. NEB Data-optimized Assembly Design (DAD): This tool designs overhang sets ...methods, such as restriction enzyme cloning, Gibson assembly, or In-Fusion cloning.  NEB Ligation Calculator...Beth Kenkel. Additional resources were added in September 2023 by Rachel Leeson.  Additional resources...

  2. The Power Behind NGS Plasmid Validation: seqWell

    Type
    Blog Post
    ...our plasmid assembly pipeline. To date, this has resulted in high quality NGS-based assemblies for over ...sequencing depth and coverage in order to, for example, assemble hundreds of different plasmids from a single Illumina...amount. plexWell™ + Addgene = high quality NGS-assembled plasmids Because of the enhanced normalization...libraries is well suited to large-scale de novo assemblies of short constructs like plasmids. The plexWell...bioinformatics pipeline that will return high-quality assembled and circularized plasmids from plexWell™ NGS data...

  3. Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection

    Type
    Blog Post
    ... calcium chloride which permeabilizes the cell membrane so the bacteria can easily uptake your plasmid...an electrical charge to cells, to increase cell membrane permeability and thus transformation efficiency...example are bacteriophages that attach to bacterial membranes and inject their genetic material into the cell... more easily cross the negatively charged cell membrane. Physical methods such as electroporation or microinjection...microinjection actually pokes holes in the cell membrane so DNA can be introduced directly into the cell...electrical pulses to create transient pores in the cell membrane that genetic material can pass through. If you...

  4. With an Eye Towards the Future, We Look Back at the March for Science

    Type
    Blog Post
    ...and numerous other reasons. It is important to remember that we, scientists and non-scientists alike, ...spoke in support of science - he's one of the few members of Congress who holds an engineering degree. It...the postdoctoral engagement committee and active member of the university's Postdoctoral Association.  ...career that could simply cease to exist. But then November hit and panic ensued as “alternative facts” became...encourages open discourse with our fellow community members. The March for Science was an important first step...available to people who want to join the dialog and 2) remember that science should not be partisan: everyone ...science communicator, UBC Public Scholar, and a member of the Vancouver SciCATs. Follow her on twitter...

  5. Special Delivery: Fluorophore Targeting for FRET Studies

    Type
    Blog Post
    ...they cannot cross cell membranes, so they are best used either with plasma membrane proteins (such as GPCRs...fluorescence wavelengths for FRET experiments. However, remember that, if you make an FP fusion, you are inserting... Most importantly, these compounds cross cell membranes, thus enabling FRET measurements in intact cells...Intact Cells Cy3NTAc Poly His  44 35-63 Plasma membrane protein, permeabilized cells  Cy5NTAc 32 25-...Intact Cells Cy3NTAc Poly His  63 50-90 Plasma membrane protein, permeabilized cells  Cy5NTAc 43 34-...Intact Cells Cy3NTAc Poly His  65 52-94 Plasma membrane protein, permeabilized cells  Cy5NTAc 59 47-...

  6. Addgene's Tips for Plasmid Quality Control

    Type
    Blog Post
    ...freely-available DNA-editing software packages to assemble your expected full plasmid sequences (examples...computer or server. This will ensure that future members of your lab can access and use the materials they...during the cloning, sample handling, or sequence assembly steps. All of our incoming plasmids are sequenced...include cloning junctions (where most sequence assembly errors occur), deletions, mutations, in-frame ...the 5’ and 3’ ends to verify that the sequence assembly and identity of the insert are correct. In other...Primer3 to help you design your custom primers. Remember, when designing a sequencing primer, you need ...

  7. Working with Nuclear Receptors

    Type
    Blog Post
    ...superfamily is comprised of 48 human and 49 murine members. NRs permit the integration and communication of...of endocrine-hormone signaling. The success of members of this group stems in part from their ability ...xenobiotic lipids derived from the diet. Additional members of this family are called orphan receptors because...coregulators exist as multiprotein complexes, a member of a single coactivator complex can serve as a ...status of SRC-3 defines its association with other members of the complex, such as p300 and CBP histone acetyltransferases...multiple cell signaling systems. Activation of membrane receptors and signaling cascades conceivably allows...

  8. Hot Plasmids and Viral Preps - March 2021

    Type
    Blog Post
    ... post: Epigenetic engineering with single-pot assembly Tracking cell-cell interactions New auxin inducible...service Epigenetic engineering with single-pot assembly By Eric Perkins Though it’s easy enough to contemplate...describes an elegant system that allows for the assembly of multiple DNA fragments efficiently and accurately... a “single pot.’   Figure 1: Single-Pot Assembly: a) Graphic depiction of chromatin within a nucleus...enzymes and reader-effector proteins. b) Golden Gate assembly (backbones available at Addgene) with examples...purposes, but the key component of the editing assemblies are sensor-actuators, either transcriptional ...

  9. Experimenting in an Open Source lab: from CRISPR to Cats

    Type
    Blog Post
    .... Since makerspaces are generally funded on a membership model - everyone contributes each month - with..., unlike your traditional biology lab, we have members with a wealth of skills to help do science on the...new replacement was not possible, but one of our members took an Arduino microcontroller and a motor controller...too expensive to make it practical - but we have members looking at how to re-use it as a platform for monitoring...fast, but with a wide range of skills among our members we’ve managed to learn a lot and cobble together... Victoria’s first hackerspace which now offers members and the public cheap access to personal manufacturing...

  10. Ligation Independent Cloning

    Type
    Protocol
    ...following T, so that there are no gaps in the final assembly. In this case, 15 bp of homologous sequence is...

  11. Centrifugation

    Type
    Protocol
    ...your lab’s instrument before use. Last Update: September 2022 Equipment Centrifuge Microfuge tubes or other...

  12. Transferable Skills Guide: Managing a Team

    Type
    Blog Post
    ... There is no way around the fact that new team members need to be trained, which may seem like a waste... a seasoned report, there should be questions! Remember that delegating is not just a way to create more...for yourself, but it will help develop your team members by giving them new opportunities. I still need...comfortable with awkward silences but I’ve learned to embrace them after one ended with the person in front of...going to get you what you really want to know. Remember that one on one discussion can be confidential...Protect your team Make you team feel safe. Your team members need to trust you and you need to encourage them...

  13. Fluorescent Proteins 101: Fluorescent Protein Timers

    Type
    Blog Post
    ...kinetics over 14 hours of expression in C. elegans embryos. However, this initial timer was a tetramer with...mammalian cells, in C. elegans, and in Xenopus embryos. Of course, timers may be sensitive to changes ... of vesicle fusion and release from the plasma membrane by fusing various vesicle cargoes to FP timer ...controlling pancreatic differentiation) in mouse embryos (Miyatsuka et al.) Distinguish between models for...fusing Medium-FT to LAMP-2A, a lysosome-associated membrane protein, and following its subcellular localization...with changes in gene expression. In a developing embryo, for example, a researcher could determine whether...

  14. Designing Your Chalk Talk for the Academic Job Interview

    Type
    Blog Post
    ...is rare these days), and a room of 5-30 faculty members. The session lasts up to an hour. You will get ...outcome with some specifics, the search committee member cannot either. Therefore, you need to be able to...idea to bounce your ideas off of senior faculty members who can help you identify overly ambitious goals...you will be interrupted frequently by committee members. As mentioned above, plan to give your 10 minute... possible to do? Research accomplishments are remembered for the problem solved, not for the amount of...faculty positions. You need to practice with faculty members that have served on search committees. If possible...

  15. Deck the Lab 2022!

    Type
    Blog Post
    ...pic.twitter.com/rExV3b8Q8u— Michael Früh (@Michelderapfel) December 19, 2022   Congratulations to all our #DeckTheLab...Borris Aldonza (보리스) 🇰🇷🇵🇭 (@PrecursorCell) December 12, 2022          View this post on Instagram... for either contest on Twitter or Instagram by December 31, 2022.  Follow @Addgene, use the hashtag #DeckTheLab...pic.twitter.com/rDaEjxTNZX— Heng Chooi (@YitHengChooi) December 4, 2021   I spent a year hoarding used tip boxes.../X4eKUGnQMe— Alex Dainis, PhD 🧬 (@AlexDainis) December 18, 2017...

  16. #DeckTheLab with Tinsel and Test Tubes, Fa La La La La La La La La

    Type
    Blog Post
    ...It’s finally December, and here at Addgene HQ in Massachusetts that means it’s time for near-freezing...decorating skills. To join the contest, simply embellish and adorn your lab space or your work from home...pic.twitter.com/OOrwo9feyJ — BTC Institute (@BTCInstitute1) December 23, 2020 Contest 2: Deck the Lab - Home Edition...pic.twitter.com/qDylF8TdHH — Matthijs Dorst (@MattNeuro) December 4, 2020 Here’s how to enter: Post your picture... for either contest on Twitter or Instagram by December 31, 2021. Let us know which contest you are entering...

  17. Five Popular Model Organisms

    Type
    Blog Post
    ...an ideal model organisms for embryonic development studies as their embryos are completely transparent...., and produce 50-300 eggs at a time. Zebrafish embryos are also laid and fertilized externally, allowing... them. Scientists can simply inject one-celled embryos with DNA or RNA to edit their genomes or to create...be easily visualized in a living organism from embryonic development to adulthood. This ability to genetically... and integration of transforming sequences." The EMBO journal 10.12 (1991): 3959-3970. PubMed PMID: 1935914...

  18. Replacing Paper: Tips for Choosing an Electronic lab Notebook

    Type
    Blog Post
    ...accessible to your team members only, under the specific permissions set for every member of your team. Do you...pricing, it is good to know how many of your lab members will use the ELN. If your lab is planning to grow..., notifications, sharing files, assigning team members to certain tasks, etc. How is security and user...to assign specific permissions to different lab members to avoid any issues later on. Do you need external...software solution that makes it much easier for lab members to manage and organize their work at every step...

  19. Multiple Plasmids at a Low Price: Kits from Addgene

    Type
    Blog Post
    ... Cell Free Protein Expression Kit FREQ Seq PUF Assembly Kit Below you’ll find information about what ...engages the ribosome for translational complex assembly. PhD student Dejan Gagoski used SITS to create...Academy of Sciences 109.52 (2012): 21408-21413. PUF Assembly Kit PUF proteins are composed of repeat segments...The Zhao lab has made this easy with their PUF Assembly Kit. The kit contains all the variant PUF segments...Sequence‐Specific Detection of RNAs in Two Colors."ChemBioChem 17.10 (2016): 895-899. Choudhury, Rajarshi,...15875. See Kendall Morgan’s blog post on the PUF Assembly Kit for some insights on its potential from its...

  22. Designer PUF Proteins for Any RNA Target

    Type
    Blog Post
    .... Get the PUF Assembly Kit! Tips and tricks For those interested to try the PUF Assembly Kit out for themselves...tool…designer PUF proteins! A newly available PUF Assembly Kit makes it possible to devise RNA binding proteins...factors (PUF). In a single step, researchers can now assemble designer PUF domains for RNA specificity engineering... Reference Abil Z, Denard CA, Zhao H.  Modular assembly of designer PUF proteins for specific post-transcriptional...

  23. FLEx Technology and Optogenetics: Flipping the switch on gene expression with high spatial and temporal resolution

    Type
    Blog Post
    ...flow of electric charges across cell membranes and maintain membrane potential in response to visible light...across the axon's membrane: once a certain number of positive ions crosses the cell membrane, a threshold ...rhodopsins enable ions to flow across the axon’s membrane, thereby controlling neural activity (Bernstein...Stoeckenius. "Rhodopsin-like protein from the purple membrane of Halobacterium halobium." Nature new biology...

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

    Type
    Blog Post
    ...also encodes the Assembly-Activating Protein (AAP) and the recently discovered Membrane Associated Accessory... AAV genome and docks with the genome onto an assembled capsid. Then Rep52/40, which also bind the capsid...generate the three VP proteins. The three VP proteins assemble into capsid at a 1:1:10 ratio (VP1:VP2:VP3). There...reading frame (ORF). While AAP promotes virion assembly, the role of MAAP has not been clearly defined...

  25. FlipGFP, a novel fluorescence protease reporter to study apoptosis

    Type
    Blog Post
    ...apoptosis reporter, ZipGFP, which utilized the self-assembling split GFP (GFP1-10 and GFP11) to increase fluorescence...animals. To do this they modified a different self-assembling split GFP (GFP1-9 and GFP10-11) by flipping a...rectify this the Shu lab first improved the self-assembling capability of sfCherry approximately 20 fold ...FlipGFP to study apoptosis in HeLa cells, zebrafish embryos, and Drosophila. They treated HeLa cells expressing...the spatiotemporal dynamics of apoptosis during embryo development. GFP fluorescence increased over time...

  26. New Neuroscience Tool: The iGluSnFR3 Glutamate Sensor

    Type
    Blog Post
    ...three different membrane-targeting flavors – PDGFR, GPI, and SGZ, to improve neuronal membrane trafficking...segment (PDGFR TMD) that anchors the sensor to the membrane and displays it on the extracellular side. In .... PDGFR is a C-terminal fusion to the PDGFR transmembrane domain in the mammalian expression pMinDisplay...glycosylphostidylinositol anchor. SGZ contains a PDGFR transmembrane domain, followed by a modified form of the cytosolic...

  27. A Deep Dive into BioBuilder

    Type
    Blog Post
    ...they hear in their classes,” she said, “but they remember the moments when they discover something new for...focused on an emerging scientific field, BioBuilder embraces new opportunities for scientific exploration, ... Lab is a fully equipped, state-of-the-art lab embedded in LabCentral’s community of innovators that opened... Blog Synthetic Biology and the Importance of Assembly Standards Synthetic Photobiology: Optogentics ...Posts Resources on Addgene.org Guide to SynBio Assembly Standards Find Plasmids for Creating and Controlling...

  28. GPCRs: How Do They Work and How Do We Study Them?

    Type
    Blog Post
    ...Receptor: GPCRs are also known as seven-(pass)-transmembrane domain receptors, due to the seven alpha helices...helices that transverse the cellular membrane. The receptor recognizes the appropriate ligand to activate...proteins on the intracellular side of the cell membrane. Heterotrimeric G proteins are important molecular...kit at Addgene! TRUPATH: Bryan Roth’s lab also assembled a collection of plasmids to elucidate the role...DREADDs) (Roth, 2016). DREADDs were engineered from members of the human muscarinic receptor family and are...

  29. CRISPR Pooled gRNA Libraries

    Type
    Collection
    ...164084 Knockout Human Sabatini 3rd 10 7,501 Human Membrane Protein Activation Library 113345 Activation Human...

  30. Open Resources and Plasmid Tools For Studying C. elegans

    Type
    Blog Post
    ...cell divisions of the C. elegans embryo when all essential embryonic genes are individually depleted via...reminded of the extent that C. elegans researchers embrace open science and share resources and tools. That...flybase. microPublication is run and maintained by members of the worm community but there are Drosophila ... lab. SapTrap is a reagent toolkit and plasmid assembly line for the creation of CRISPR targeting vectors...

  31. The Scientific Conference Poster Session: Tips for Success

    Type
    Blog Post
    ...fundamental takeaway. Writing these points will help you remember what to say and provide your audience with a clear...venue and professionalism of the conference, and remember to wear comfortable shoes, as you will likely ...your breath before diving in to the next figure. Remember your outline: Instead of getting bogged down in... the minutia or coming off as a broken record, remember the outline you created for yourself. Hit the ... Resources on the Addgene website Become of a member of the Addgene depositor community Learn about ...

  32. Hot Plasmids: Winter 2025

    Type
    Blog Post
    ...Cultured rat hippocampal neuron expressing pan-membrane ASAP5. Solid blue circle marks the initiation ...via the Kv2.1 targeting motif as well as a pan-membrane construct without the Kv motif. Altogether, if...kinetics and high responsivity near the resting membrane potential, ASAP5 is a great new option. Find ASAP5...specifically binds to active (GTP-bound) Gα. Gα-YFP assembles into functional heterotrimers with endogenous ...optional accessory plasmids, the collection targets members of all four G protein families: Gs, Gi/o, Gq/11...

  33. What's Your Organism? Expanding Genomic Tools via the NSF EDGE Program

    Type
    Blog Post
    ...pic.twitter.com/sIIoIsIFd1 — Henry Lab (@EvoEmbryo) September 24, 2018   Xenopus tropicalis (Gary Gorbsky..., mix them in a tank, and observe the beauty of embryo development in real time. I was already a biology...nucleus and put it in a fertilized egg to get edited embryos (replacing the nucleus is still very difficult)...female mosquitoes. Now they are trying to replace embryo injection (hard to do, expensive, not every lab...

  34. Bioinformatics at Addgene

    Type
    Blog Post
    ...start using. Prior experimenting with open source assembly algorithms meant that we already had a clear idea... individual steps to automate plasmid sequence assembly, and possible solutions for each in the form of...quality score analysis, k-mer counting, or sequence assembly), but the order and usage of the scripts needs...the freedom to use multiple open-source sequence assembly tools at the appropriate place in our workflow...Tower to augment our pipeline and enable our team members to automate their weekly sequencing tasks. More...

  35. Transferable Skills Guide: Creativity

    Type
    Blog Post
    ...Anyone could fill out the form at any time and a member of our Business Development team would review it.... Identify your enemies In Felicia Day’s book Embrace your Weird, Day identifies nine enemies that stand...Frida Kahlo or you’re stuck drawing stick figures, remember your training as a scientist has been building...world. Penguin Publishing Group. Day, F. (2019). Embrace your weird face your fears and unleash creativity...creativity. Gallery Books. Boyes, A. (2019, November 13). 5 mental mistakes that kill your productivity. Harvard...

  36. How to Be an Excellent Trainee

    Type
    Blog Post
    ... place, learning something new. So how can you remember everything? Write it all down! And I mean everything...second rack in the corner freezer sounds easy to remember in the moment….but you’ll probably forget and ...going to each space in order to double-check you remember where the equipment, reagents, and samples are...to ask! Especially for those new to the bench, remember to let your bench mentor know what you don’t know...polite, appreciative, and clear about their needs. Remember, your bench mentor’s training time is time set...

  37. Plasmids 101: Stringent Regulation of Replication

    Type
    Blog Post
    ...specific RNA site. The EMBO Journal, 17(17), 5201–5213. https://doi.org/10.1093/emboj/17.17.5201 del Solar...plasmid copy number. The EMBO Journal, 20(24), 7323–7332. https://doi.org/10.1093/emboj/20.24.7323 Resources...post was originally written by Jason Niehaus in December 2015 and was updated by Emily P. Bentley in June...

  38. Lentivirus Production

    Type
    Protocol
    ...drifted. Filter the solution through a 0.22 μm membrane. Aliquot 500–1000 μL into sterile tubes. Store...

  39. Five Popular Model Organisms, Part 2

    Type
    Blog Post
    ...and breed and they have large, easily modifiable embryos, which is ideal for observing and manipulating ...majority of studies involving axolotls focus on embryonics and biochemistry. Though limited at first, advances...humans so they are often used to study vertebrate embryology and cell development. One major resource for ...understand human disease alleles during and after embryogenesis. Addgene has over 600 plasmids that contain ... a Model Organism. News-Medical. Retrieved on September 18, 2020 from https://www.news-medical.net/life-sciences...

  40. Four Ways to Package Transgenes That Exceed the Size Limit of Adeno-associated Virus

    Type
    Blog Post
    ...By breaking the transgene into smaller pieces. Reassembly of fragmented genomes is the key to fitting large...full-expression cassette. AAV’s natural ability to reassemble fragmented genomes is the basis of several split...with both part A and part B AAVs, the fragments reassemble and produce the full-length gene. Below are overviews...Vector, Big Gene Transduction: Fragmented Genome Reassembly of Adeno-associated Virus. Molecular Therapy ...of large genes to the retina by dual AAV vectors. EMBO Mol Med 6:194–211 . https://doi.org/10.1002/emmm...
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