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

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

...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 ...

...frozen bacteria, preventing damage to the cell membranes and keeping the cells alive. A glycerol stock ...

...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...

...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...

... 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...

...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...

... 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...

...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...

...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...

...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...

...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...

...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 ...

...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 ...

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

...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...

...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...

... 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...

...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-...

...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...

...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 ...

...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...

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

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

... 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 ...

.... 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...

... 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...

...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...

...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...

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

...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...

...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...

...using fluorescence microscopy. Last reviewed on: September 9, 2023...

...the chemical hygiene officer. BSL-2 Guidelines Remember, the BSL-1 laboratory guidelines above are expected...

...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...

...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...

... 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...

.... 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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

...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 ...

...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...

...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...

...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...