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...research program (think a subtle difference in mouse chow, a different water filtration system, environmental...outline and refer back to the original paper that everyone in your lab has been referencing for the last...original researchers actually did. Admittedly, the differences are often very subtle, and it can take a lot ...and it was just that they were doing something differently. Communication comes in many forms—from transparency...other publishers and organizations have various different versions of checklists and guidelines to help ...rewarding good scientific citizenship in all its different forms as part of graduation, hiring, funding, ...

...cells (Ma et al., 2015). To do so, they fused a different fluorescent protein to various Cas9 variants, ...either MS2, PP7, or boxB) that each recruits a different fluorescent protein (BFP, GFP, or RFP) to the ...green, or red readout. But gRNAs containing two different hairpins will produce a secondary color (yellow...allows for simultaneous visualization of sites on different chromosomes in living cells, the system requires...they chose an intergenic DNA region (IDR3) as a reference loci to measure inter-locus distances of loci ...sgRNA-Sirius-4X(MS2-PP7) showing recruitment of different fluorescent proteins. (B) CRISPR-Sirius used to...try. Find CRISPR-Sirius plasmids at Addgene. References Ma, Hanhui et al. “Multicolor CRISPR labeling...

...not exert force by dynamically rearranging into different polymerization states. Instead, R body monomers...natural context or 3) be modified to extend at different pH levels. Domesticating R bodies We have shown...that R bodies can function to break membranes of different organisms. And finally, we demonstrated that R... can be “tuned” with mutagenesis to extend at different pH values. Using a screen that takes advantage... fact that coiled R-bodies sediment in certain buffers but remain soluble when extended (see figure to...region produced R-bodies that could extend at different pHs. Plasmids expressing expressing both wild ...School. Follow her on Twitter @jessicapolka.     References 1. Kanabrocki, J. A., Quackenbush, R. L. & Pond...

... labs (see table below) have different PAM requirements. These different PAM requirements allow researchers...discovered all the time and the requirements of these different systems can vary (for example, xCas9 is a variant...neither bind nor cut the target. Cas9 homologs from different organisms or Cas9 mutants developed in a variety...researchers to target many different genomic loci. 3. Cleave target DNA resulting in a double-strand break...). Throughout the remainder of this post, we’ll refer to this RNA complex as the “gRNA”. The gRNA contains...choice (see tips for designing a gRNA) and their prefered Cas9 variant (with the appropriate PAM sequence...sequence.   Now that you know about all the different components of CRISPR, It's time to start thinking...

...student or postdoc may require different types of mentoring. Some mentees prefer lots of advice and micromanagement...an effective working environment? Tom Ellis was offered a start-up package that included a postdoc and ...recommends to seek out advice from seasoned PIs who can offer support. “It is an exciting and emotional time!”...recruit people who are nice as top criterion and offers advice to not rush when growing your lab. “One ... lab and safety manager, researcher, adviser, conference planner, and writer.” Connie Cepko was surprised...impossible' or useless.” How do you run your lab differently or the same as your adviser did? Tom Ellis merges...online. The Howard Hughes Medical Institute (HHMI) offers some great resources for Early Career Scientists...

...libraries expand upon the CRISPR family of plasmids by offering a lentivirus-based mechanism for sgRNA delivery... microscope 8 hours post-pulse. The distinct differences of red-to-green fluorescence in individual mitochondria...mitochondria in the networks marks the differences in import of newly synthesized (green) protein and ...five previously unknown channelrhodopsins from different species: Stigleoclonium helveticum (ShChR), Chlamydomonas...advances, the Ting lab’s first-generation APEX suffered from inconsistent activity levels and limited ...cassette. They validated this methodology in two differents types of bacteria (E. coli and Salmonella typhimurium...naturally-occurring variations in the TALE repeat, referred to as “non-RVD variations”, were found to improve...

...2025 and updated in August 2025. References and resources References Ahuja, V., Jeyabalan, S., & Tzanakakis...multiple coreceptors, which may be present on different cell types and sometimes vary between target species... numbers separated by a slash: the first number refers to the genome, and the second number to the capsid...AAV8, AAV9 In addition to tissue tropism, different AAV serotypes can be useful to evade neutralizing... (AAV) Serotype 9 Provides Global Cardiac Gene Transfer Superior to AAV1, AAV6, AAV7, and AAV8 in the ...Hemagglutination and Efficient Transduction but Differ in Sialic Acid Linkage Specificity. Journal of ... Dudman, J. T., Hantman, A. W., Looger, L. L., Schaffer, D. V., & Karpova, A. Y. (2016). A Designer AAV...

...check out these blog posts.       References and resources References Burry, Richard W. “Controls for ..., you will have a better understanding of the different types of controls and be able to use this information...treatment versus and which are likely just natural differences between individuals. Recommended controls for...extremely bright, then the noise likely will not interfere. If the fluorescence from your target is weak,...fluorescence of the sample, or autofluorescence, also interferes with data interpretation and can be addressed....  Western blotting When comparing samples in different lanes in a western blot, you’ll need to include...equal loading across samples and equal protein transfer from the gel to the membrane. A loading control...

...plasmid sample has a mix of different-sized multimers, you may even see different sets of bands on the gel... us at [email protected]. Resources and References References Bacolla A, Wang G, Jain A, Chuzhanova NA...Nanopore sequencing or PacBio SMRT sequencing, (which differ from the short-read Illumina MiSeq sequencing that...multimerization has been observed to occur in many different kinds of plasmids, taking many forms (Levene, ...from BiteSize Bio for more information on how different plasmid conformations run on an agarose gel). ...low metabolic burden seed-stock and fed-batch fermentation processes. Biotechnol Bioeng. 2009 Aug 15;103...Plasmids 101 eBookAddgene's Molecular Biology ReferenceAddgene's Restriction Digest Protocol More resources...

...isolated from different organisms and engineered Cas9 variants that were able to target different PAM sites...activity of Sniper-Cas9 to different Cas9 variants using gRNAs of different lengths (Lee et al., 2018)...found in other species can recognize a variety of different PAM sequences. Researchers have also used experimental...activity relative to Cas9. Since then, the Joung and Jennifer Doudna Labs used targeted mutagenesis to generate...relative to eSpCas9 and SpCas9-HF across a number of different sites. Using a combined random and directed mutagenesis...choosing a Cas protein for your experiment. References Hu, Johnny H., et al. "Evolved Cas9 variants ...

... the best method for titering AAV? Is there a difference in titer results between ddPCR and qPCR? A: At...curve and somewhat more reliable quantitation. Differences in sample treatment across methods can affect...  Q5: What is the efficiency of co-delivering different AAV particles into the same cells in vivo? A: ...give some insight into codelivery efficiency at different doses. See Figure 4 in this paper for more information...very high dosage. Any thoughts?  A: The question refers to this paper. AAV can be safely concentrated to...IACUC committee.  Animal Models Q9: Is there any difference in transfection efficiency when delivering these...injections? A: We have not observed any appreciable difference in AAV transduction efficiency between retro-...

... the retina using viral tools.   References and Resources References Pillay, S., et al. "An essential ...and can be specifically targeted to a number of different cell types even when systemically administered...researchers speak about AAVs, they are actually referring to rAAVs. ITRs and serotypes The names of AAVs...ITRs, which comes from the AAV2 serotype. Serotype refers to the type of capsid (outer protein shell) used...capsids allow the virus to infect (or not infect) different cell types with some specificity. There are a ...will lose expression quicker than terminally differentiated, non-dividing cells.   Figure 2: ...subretinal injections of neonatal mouse eyes at two different ages." Retinal Degenerative Diseases. Springer...

... 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...

...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...

...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...

... 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 ...

...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...

...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...

...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...

...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. “...

...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...

...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...

...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....

...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,...

...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 ...

... 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...

...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 ...

...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...

...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 ...

...tools with a focus on plants.     References and Resources References Karmakar, S., Panda, D., Baig, ...TnpB protein, much like how PAM sites change for different Cas species. For instance, the TnpB from Deinococcus...Deinococcus geothermalis (Dge10TnpB) targets a different TAM (5′-TTAT-3′). The unique TAM requirements ... genomic sites that can be targeted with TnpB, offering access to regions previously unreachable with ...commonly used nucleases like Cas9. This is where TnpB offers a clear advantage. At approximately one-third the...for plant genome editing using TnpB. The main difference between them lies in the choice of promoter for...Engineering the Plant Genome Using CRISPR/Cas9 RNA Interference in Plant Biology: New Tools for an Old Favorite...

...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...

...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...