... and offer educated insight into what the desirable features of future technologies should offer in order... funding towards my PhD project. Grant writing offers various skill building opportunities that graduate... to a given problem, Empowers the writer, And offers insight into a career as a Professor. Step ...Challenge motivates the request for application and offers an opportunity to summarize the limitations of ...potential pitfalls of the intended protocol, and offers alternative solutions to demonstrate thorough thought...standard “Our work will empower the world” tone that differs from publication writing and may be challenging...and exposure, here are some additional links that offer more advice on writing a grant proposal. "Murder...

...Figure 9: epegRNAs (left) and PE7 (right) offer two different strategies for protecting pegRNAs from degradation...software in the comments below! References and Resources References Anzalone, A. V., Randolph, P. B.,... These tips are a great starting point: Test different lengths of the primer binding site, starting with...still be optimized (Anzalone et al., 2019). Test different lengths of reverse transcriptase template, starting... templates, it is even more important to test different lengths and sequences, as unintended secondary...specific prime editing tools There are a lot of different prime editing tools to choose from, and many have...

...you can quickly test that part in different contexts and plug different characterized parts into a TU! What...works on genetic code expansion.  References and Resources References Weber E, Engler C, Gruetzner R, Werner...cloning is MoClo. That’s because MoClo specifically refers to the organizational schema described above, and...nature of MoClo allows users to test a variety of different orientations and components within the system ... a great tool to deliver many combinations of different candidate genes at once to identify the best cocktail...and gene editing that use MoClo, for which many different MoClo kits have already been established. Keep...

...tools for genetic engineering. There are several different viral vectors to choose from, for example gamma-retrovirus...separate envelope and packaging plasmids as well as transfer plasmids. This concept ensures safe handling and...isoforms of these proteins, as well as the viral LTRs differ. Therefore, lentiviral and gamma-retroviral packaging...Gamma-retroviral vectors Popular gamma-Retroviral Transfer Vectors   Expressing your gene of interest pMKO...expressing RNAi pMKO.1 puro (Plasmid #8452) RNA interference (RNAi) is an RNA-mediated gene silencing mechanism...interest. Lentiviral vectors Popular Lentiviral Transfer Vectors    Expressing your gene of interest pLenti-puro... in phenotype. The plasmid comes with a 1.9 kb stuffer sequence that can be removed and replaced with ...

...pathological conditions. References: Monticelli, M., Ferro, T., Jaeken, J., dos Reis Ferreira, V. & Videira, P...GdnHCl to patients suffering from rare diseases arising from Arg mutations. References: Baldwin, Enoch,...life-threatening consequences. They result from defects in different steps in the glycan biosynthetic pathway and have...recently reviewed and reported that PMM2-CDG patients suffer from immunological defects. These are characterized... discovered that the in vitro functions of 11 different Arg-mutant enzymes could be rescued by supplementation...

... can only carry around 4.5 kb. Different AAV serotypes have preferences for specific cell types. For example...behavioral studies. References Stoica L, Ahmed SS, Gao G, Sena-Esteves M. Gene transfer to the CNS using ...the experiment. Handling can however negatively interfere with certain tests and is not always recommended...end of an experiment series would behave very differently (distracted) from the ones tested at the beginning...Experiment Behavioral scoring systems (experimenter differences in behavior observation); experimenter blinding...injections. AAV particles are smaller and may be preferable if wider diffusion is required. On the other ...

...never been so important.     References and resources References Biering SB,  Sarnik SA, Wang E, Zengel... study the mechanisms of infection and how to interfere with it in a BSL-2 lab instead of a BSL-3 lab....for generating pseudovirus and those encoding different spike protein variants that can be incorporated...vaccinated individuals are at neutralizing the different SARS-CoV-2 spike variants. You can learn more ...widely used qPCR and antigen tests.  Labs including Jennifer Doudna’s lab and Feng Zheng’s lab had already ...head over to this blog post that highlights five different methods to use CRISPR to detect SARS-CoV-2. Other... Jennifer Tsang...

...publish a new blog post, and live tweet from conferences. Find more tips on using social media as a scientist...for example at an anniversary or for Girls’ Day, offer guided tours to show people where the lab magic ...communicate science. You can draw live sketches at conferences like @ATJCagan or create agar art with colorful...Association, EUSJA or ABSW for helpful tips and job offers. If you live in the UK, join the SciComm mailing...10 days. And the Society of Experimental Biology offers press internships to cover their annual meetings...College London. Similarly, the University of Cardiff offers an MSc in Science, Media and Communication. In ...Santa Cruz, MIT, UW Madison, and Boston University offer science communication programs. Others, like Northwestern...

...collaborate with specialists in different fields that can provide different insight into an area - not just...you are looking to move to a different university, perhaps in a different country, choose where you go...collaborators It’s a good idea to move around to different labs during your career; you learn new techniques...I hope to discover. This section may make the difference in whether you get funding or not; applications... grants too There are actually quite a lot of different pots of money available for researchers, so look...gained in writing small travel grants to attend conferences and a seed funding grant for a proof-of-principal...

...these will not be detailed here, but additional references are provided at the end.  Nearly all common bacterial..., and III), each responsible for transcribing different types of RNA: Polymerase I is responsible for ...that each eukaryotic RNA polymerase terminates differently. Polymerase III, for example, relies on a specific...Rho-independent termination found in prokaryotes. This is different than Polymerases I and II, which both require ...factor dependent, Polymerases I and II employ different mechanisms to terminate transcription. Polymerase...and/or use weaker poly(A) signals like BGH.  References Peters JM, Vangeloff AD, Landick R. Bacterial...Promoter-Dependent Manner. Hum Gene Ther. 2008. PMID: 18627247 Zufferey R, Donello JE, Trono D, Hope TJ. Woodchuck Hepatitis...

... optical insulator called a ferrule (below). The fiber optic cable/ferrule is inserted into the target...optic patch cable is then connected from laser to ferrule to deliver light pulses to the target brain region...Several protocols have been written outlining the different steps and materials required for each [1, 2]. ...photons from the laser to the patch cable to the ferrule implant must be maintained during the entire protocol...motor cortex of a mouse. Upon connection of the ferrule implant to the laser with the patch cable (difficult...discussed above, each opsin variant has slightly different activation/inactivation kinetics. For example,... or follow him on Twitter @derekpsimonphd.    References 1. Gutierrez DV, et al. Optogenetic control of...

...yours does and then use that time! Different platforms have different quirks, so remember to practice on...that requires its students to do rotations in different labs before selecting an advisor, it is always...! Prepping for the interview Many universities offer free practice sessions for students doing virtual...points and keeping them at-hand, you can quickly reference your questions about the PI’s research or the ...the name of the award you won for your conference presentation. It can help soothe your nerves and make ...like sickness or scheduling issues can and do interfere with those in-person plans. Virtual options are...good fit for you. The application process looks different than it did a few years ago, but I hope the tips...

...advantages SapTrap offers for tagging a single genetic locus, the toolkit also offers repair templates ...improve CRISPR genomic tagging in C. elegans. SapTrap offers scientists a convenient one tube assembly reaction...will produce the same unique SapI 5’ overhang, different combinations of connector and tag donor plasmids...donor plasmids for protein tagging, including luciferase (NanoLuc), fluorescent proteins (TagGFP2, TagBFP...cell line stably expressing Cas9. This PCR toolkit offers C- and N-terminal tagging vectors (eg GFP, Flag...protein expression levels. CRISPR tagging systems offer users an easy and efficient way to tag endogenous...light on previously unknown protein functions. References Schwartz, Matthew L. and Eric M. Jorgensen. “...

...extraordinary molecular detail.   References 1.Patterson, George H., and Jennifer Lippincott-Schwartz. "A photoactivatable...Fluorescent proteins (FPs) offer scientists a simple yet powerful way to tag cellular proteins and investigate...most scientists working on in vivo imaging models perfer the PA-RFPs over PA-GFP. Another class of irreversible...dimeric or tandem dimer versions of EosFP are preferred over the monomeric one since the formation of ...organelle trafficking. Monomeric PA-FPs are usually preferred for studying single protein characteristics, as... as the monomeric state is less likely to interfere with endogenous protein structure and function. By...non-invasive labelling, but in contrast, PA-FPs offer a reduced potential for photobleaching and phototoxicity...

...most cases, interested in transferring genes to mammalian cells (i.e. transferring DNA), yet retroviruses...lentiviral vectors. Lentiviruses are sometimes referred to as “complex retroviruses ” (Cullen, 1992) because...generalize the idea of “transduction” to mean any transfer of material from the virus to the host. In fact...pseudotransduction” — to describe the direct protein transfer of RT, which would actually occur in step 2 during...dimerized linear ssRNA strands (which can have a different genomic sequence from one another) — by various... with lentiviral replication.  Resources and References More resources on the Addgene blog Viral Vectors...popular lentiviral plasmids γ-Retrovirus Guide References Cullen, B. R. (1992). Mechanism of action of ...

...helping everybody, helps everybody! References and resources References Dayton, R. D., Grames, M. S., & ...scientists under-informed with respect to how different serotypes, promoters, and injection methods are...published.    Fig. 1: Testing of three different AAV serotypes (AAV1, 8, 9) injected into the A2...biological reagents; many factors, like storage buffers and conditions (3,5,8), injection titers (1,2),...literature, scientists can use the Data Hub to compare different methods and vectors, and use this data to guide...& Klein, R. L. (2018). More expansive gene transfer to the rat CNS: AAV PHP.EB vector dose–response and...sharing practices and data availability upon request differ across scientific disciplines. Scientific Data,...

...to the plasmid search results: More than 200 different GCaMP containing plasmids are available for distribution...biosensor using AAV, you might decide you’d really prefer to order ready-to-use virus rather than producing...starting a new search or clicking through to a different part of the website, you can now use the pulldown...pulldown menu on the search page to quickly jump to different types of items: Did you notice the “Filter By.... The following screenshots show the range of different terms we can use to find materials from this publication...dropdown menus below the search bar. As you try different terms you may find the available categories and...you get this far and realize you need to apply different filters you do not need to start a new search....

... number of different strains of E. coli that can be used for transformation. The preferred E. coli strains...freezers that the samples can be stored in? Different shelves? Different racks on each shelf? Give each container...long-term storage in our facilities, every sample is transferred to a 2D barcoded tube (see next section) and ...about location, location, location There are many different types of samples at Addgene - DNA sent from depositors... concentration or need to be transformed into different cells than were originally requested. Most of ...analysis from our protocols and molecular biology reference pages! Resources Avaliable on the Addgene Blog...

... protein as pure as possible? Vectors that offer different combinations of tags are readily available,...peptide-based proteosome inhibitors, glutathione S-transferase (GST), which can be fused to recombinant proteins...Purification Binding and elution steps use very moderate buffer conditions FLAG DYKDDDD or DYKDDDDK or DYKDDDK...co-IP, IF, flow -cytometry; can occassionally interfere with protein folding HBH HHHHHHAGKA GEGEIPAPLA...small size, rarely affects function S-tag KETAAAKFERQHMDS  1.8 Solubility and Affinity Abundance of charged...common. Tandem affinity purification (TAP) once referred specifically to a combo tag comprised of a calmodulin...information and some protocols can be found in the references provided. Additional resources: Young CL, Britton...

...made it possible to offer DayCon as a free event! Organizing a science conference for the public How ...first-time event called DayCon. DayCon is a one-day conference aimed at the general public that provides an ...graduate student at Harvard, who wanted to provide a conference-style experience that was accessible to the general...How does one organize a public scientific conference? As a member of the DayCon Executive Board, I was fortunate...strength in the biomedical sciences and decided to offer a varied format event to keep the audience engaged... make DayCon a yearly event and to expand the conference to accommodate a larger number of attendees. ...

...Vector Core. Transfections are performed using the transfer plasmid, a plasmid encoding rep and serotype-specific...taken offline and discarded. Confirmation of the transfer plasmid Addgene uses a rigorous barcode matching...preparation undergoes PCR with primers targeting the transfer plasmid used in the transfection. PCR products... matching system and PCR-based confirmation of transfer plasmids used in Addgene’s viral production. Barcoding...After production, we confirm the identity of the transfer plasmid in the viral preparation by performing...performing PCR against unique regions of the transfer plasmid.   Lentivirus resources Browse lentiviral preps... by Ina Ersing in August 2023. Resources and references Resources at the Addgene blog Find and share ...

...immunotherapy than ever before. Although different types of cancer have different challenges and obstacles to overcome...However, there are many different forms of immunotherapy that work in different ways. For example, a new...learned much about the causes of cancer and the different avenues that can be used to treat it. For those...overcome, mesothelioma sufferers can see great promise in up and coming treatments like immunotherapy....latency period, and rarity of mesothelioma make it different from many other types of cancer. Mesothelioma ...mesothelioma, such as in the case of Paul Zygielbaum. A different approach to immunotherapy is bevacizumab (Avastin...

...husbandry and ability to produce abundant, externally fertilized, transparent progeny (Figure 2). These features...regulate processes from development to metabolism to fertility. Several zebrafish-specific gene expression databases...generating mutants. Unexpectedly, phenotypes sometimes differ depending on whether a gene is deactivated via ...researchers to observe changes to these tissues under different experimental conditions. Strategies for generating...tracing Over a period of 24 hours, the externally fertilized zebrafish zygote transforms into an organism ...sequencing approaches (Harland, 2018). Several different methods have been developed, relying on established...vertebratedevelopment using optogenetic approaches.     References El-Brolosy, Mohamed A., et al. "Genetic compensation...

...opportunity to discover and evolve your transferable skills. Transferable skills are the non-technical skills...graduate student at a networking event recently who referred to her lab as her family three times during a ...formulate hypotheses and conduct experiments. You transfer liquids between tubes, look through microscopes...etc.--so everyone’s experience will be a little different. Some of these changes you may enjoy immediately...production, I didn’t really start to recognize my transferable (or “soft”) skills until I was out of the lab...lab to think about these--get started with our Transferable Skills Guide.     Over the last decade, my... blog Visit our careers topic page Read our transferable skills guide Read blog posts about career readiness...

...translation of an array of different reporters including mScarlet-I, luciferase, antibiotic resistance markers...fluorescent proteins, antibiotic resistance, and luciferase. References Gaudelli NM, Komor AC, Rees HA, Packer...et al., 2018. APOBEC-Cas9n-UGI targets its preferred trinucleotide motifs (5’-TCA-to-TUA) found within...using FACS which could limit their use across different cell types and systems. To introduce flexibility... GO works by affecting protein translation of different reporter proteins. Protein translation requires...Reporters in Gene On include a mScarlet reporter, luciferase reporter, and neomycin resistance reporter. Image...

...lead to wildly different ideas of how to approach a problem, but it is these very differences that make our...the software design. Each of us brings slightly different cognitive biases as a result of our diversity ...Addgene and ourselves. We are also able to consider different technical approaches that may come up during discussion...developer and I get to see an approach that is likely different from what I would have done. And finally the stakeholder...isn’t how we work. We all take turns working on different parts of the code. I think the best way to learn...shipping sprint and all the stories will touch different parts of the shipping code. The following sprint...every part of the software, we are able to get different perspectives on how things are implemented and...

...Bioluminescence Resonance Energy Transfer CRET= Chemiluminescence Resonance Energy Transfer    Interesting Articles...FRET, aka Fluorescence (Förster) Resonance Energy Transfer, technique which allows the detection of molecules...distance-dependent physical process by which energy is transferred non-radiatively from an excited molecular fluorophore...FRET experiments. You may have to design several different constructs to find one that could be used for ...higher than the signal generated by the energy transfer. On the contrary, if you choose a pair that are...cross-talk but you also don’t have any resonance transfer. You have to find a balance between FRET efficiency...RB, Periasamy A. Fluorescence resonance energy transfer (FRET) microscopy imaging of live cell protein...

...between Cas9 enzymes from different species, explaining why they recognize different guide RNAs. Deep in the...Other CRISPR enzymes like Cas12 have different structures and different uses. In all cases, structural biology...techniques, like electron microscopy and FRET, can offer more information on these regions.   Cas9’s transition...from the same angle to highlight conformational differences. HNH, red; RuvC, orange; CTD, yellow; REC lobe...research. New details are still being uncovered, and different techniques all bring their own advantages—and ...Allergy and Infectious Diseases. Resources and references More resources on addgene.org  Addgene's CRISPR...656. https://doi.org/10.1038/s41579-022-00739-4 References Bravo, J. P. K., Liu, M.-S., Hibshman, G. N.,...

...delivered on a different plasmid and allows simultaneous multiplex editing of six different genes. This ... Jan 28, 2016 and last updated Sep 10, 2020 by Jennifer Tsang. CRISPR makes it easy to target multiple...flanking the promoter-gRNA construct, but with different sequences adjacent to the sites. When digested...assembly principles, but they’re optimized for different purposes.   Gersbach Lab multiplexing plasmids...kanamycin-resistant plasmids, each containing a different gRNA target sequence downstream of the 7SK, human...users to express up to 7 gRNAs. The kit contains different destination vectors depending on the total number...transcript. Rather than being transcribed from different promoters, the gRNAs are transcribed together ...

...is to offer plasmids created as part of research projects, another way is to systematically offer thousands...worked closely with the legal partners and Tech Transfer Offices at universities to make COVID-19 research-related...United States, and many universities and Technology Transfer Offices were, at least, temporarily closing, these...scientists, we’ve worked with many scientists, tech transfer offices, research foundations, and funding initiatives...them available to industry, please reach out at techtransfer@addgene.org and we will initiate a conversation...conversation with you and your technology transfer office to discuss the possibilities for expanded sharing. ...

...written by Jennifer Tsang in October 2019 and updated by Emily P. Bentley in December 2024. References and ...(Anzalone et al., 2019). Plus, it can combine different types of edits with one another. All of this is...editing window cannot edit within what Fyodor Urnov refers to as “PAM deserts” in the genome. More versatile... instances where traditional base editors are preferred. For instance, if target nucleotides are positioned...type, and goal of the experiment can all impose different constraints and affect how you select and design...5.1-fold (though up to 45-fold) higher across different genomic sites compared to PE1 (Anzalone et al....used with any of the PE2-5 approaches, sometimes referred to as PE2max, PE3max, etc. PE6: Specialized prime...

...by Addgenie Rebecca Hutcheson.   References and resources References Banasik, M. B., & McCray, P. B., ...second-generation system as an example.  The lentiviral transfer plasmid encodes your insert of interest, flanked...then, numerous variants have been developed with different mechanisms to inactivate the integrase.  The IDLV...vectors is random, and the integration site will be different for each transduced cell, there’s no telling which... that of regular lentiviral vectors. The only difference is that you would use the integrase-deficient...that titering these IDLVs may require a slightly different protocol than regular lentiviral vectors. This...quantifying the integrated viral DNA along with a reference cellular gene. When titering IDLVs, there is no...

..., protected samples and was compatible with the buffer additive MgCl2 and the crosslinking agent glutaraldehyde...PEs) via engineered virus-like particles (eVLPs) offers an option to transduce cells directly with ribonucleoprotein...editor consisting of dCas9 fused to a DNA methyltransferase — that can drive lasting transcriptional silencing...Histone tail for Autoinhibition Release of Methyltransferase (CHARM), with several advantages over the ...  Rather than overexpressing an exogenous methyltransferase for epigenetic silencing, which can be toxic...tail to recruit and stimulate the endogenous methyltransferase. The strategy is generalizable to a variety...D3L recruits the endogenous, autoinhibited methyltransferase DNMT3A to the genomic location specified by...

...sensors with different ITAM/SH2 reporter pairs allow multiplexed measurement of different RTKs. Although...response. They generated variants optimized for different applications (v82 has higher affinity for glutamate...Measuring local temperature variations and heat transfer at the subcellular level requires a probe with...By fusing two bright fluorescent proteins with different thermal quenching rates (mNeonGreen and tdTomato...sub-millisecond resolution and sub-degree temperature differences under a variety of imaging conditions (155 μs...fast measurement of temperature and local heat transfer. Image reproduced from Lu et al. 2022, under CC-BY-NC-ND...plasmids here!   Lu K, et al. Intracellular Heat Transfer and Thermal Property Revealed by Kilohertz Temperature...

...Addgene, I was about to have a check-in with my boss Jennifer. I had finally found my footing with the company... for what we thought would be a couple months. Jennifer and I spent about an hour that morning scrambling... involved multiple people in tight locations. Conference rooms for on-screen presenters! Labs and benches...know cameras, but we needed someone else. Design: Jennifer thankfully volunteered, and we decided to focus...action would be to use an iPad to shoot.  Build: Jennifer created a mobile camera area that included iPad...Zoom, everyone’s new favorite third-party video conferencing software. Interviewees could simply boot up ...while the camera is here, the operator is in a different room.   Design: The previous cycle made me...

...solubility, and thus different reagents are useful for biotinylation in different microenvironments. There...used today in molecular biology for an array of different techniques and protocols. In this post we will...and decarboxylases. These enzymes catalyze the transfer of carboxyl groups to organic acids to produce...relatively small, water soluble molecule that does not interfere with the macromolecules it is added to. Biotin... signal and easier detection. This strategy is referred to as the Avidin-Biotin Complex (ABC) immunohistochemistry...variety of biotinylation reagents available that differ in the functional groups or residues they target...a solid substrate (Holden and Cremer, 2003). References 1. Chapman-Smith, A., & Cronan, J. E. “Molecular...

...scientists have developed and fine tuned many different ways to clone DNA fragments which have provided...more and more common, and for good reason. They offer many advantages over the traditional restriction...with an enzyme master mix, which contains three different enzymes: an exonuclease, which chews back the... this doesn’t allow for mix and match of many different fragments. One way around this is to use a combination...kit you will be using. Some of these products may offer advantages that Gibson assembly does not such as...with a G!) but actually work by significantly different methods. Golden Gate cloning does result in the...from one vector to another. There are lots of different ways to clone these days. Gibson and the other...

...are communicating across disciplines or doing different tasks. But these challenges can be exaggerated...equal opportunity to contribute. Using video conferencing tools like Zoom or Google Hangouts is a great...great way to include researchers from different locations. Having virtual face-to-face meetings as opposed... collaboration includes scientists working in different time zones, it’s important that everyone can access...project at the International Cancer Immunotherapy Conference in Frankfurt, Germany When funding and circumstances...techniques are being performed the same way at different sites. I did a lab exchange as part of the collaboration...been able to do from my home lab. Scientific conferences and workshops are another great place to meet...

...system which has been designed to perform gene transfer in vertebrates. The system is comprised of 2 components...transposase has been discovered in the genomes of many different fish species in a variety of non-functional forms...the lab showed that efficiencies of stable gene transfer using SB100x were comparable to stable transduction... vectors were developed into 16 variants with different selection markers (blasticidin, neomycin, puromycin... each contain two SfiI restriction sites with different overhangs depending upon whether they are on the...only likely to grow in the coming decades.  References 1. Kowarz E, et al. Optimized Sleeping Beauty... Beauty transposase enables robust stable gene transfer in vertebrates. Nat Genet. 2009 Jun;41(6):753-...

...out if you want to learn more. References and resources References Balloux, F., & van Dorp, L. (2017...called an envelope. Viruses without an envelope are referred to as naked or non-enveloped, while viruses with...viruses, here’s a quick tip on terminology. “Virus” refers to a single type of virus, not the individual viral...infection — researchers were able to create far safer viral vectors that can be used in BSL-1 or BSL-2...experimentally controlled conditions, making them far safer to work with than virions. The four types of viral...vectors (which many researchers will informally refer to as viruses) are named after the viruses they’...