...human cell lines. Nucleic Acids Research. 2014;43(3):e17. doi:10.1093/nar/gku1198... to devise an experiment that can tell you more about its function.  Resources and references Resources...

... covered. Happy voltage sensing!   Figure 3: Components of the types of voltage indicators reviewed...especially essential. If you want to learn more about how viruses can be used for chemigenetics, biosensors...

...European ancestry.  Understanding the impact Fig. 3: Histograms indicating the frequency (x-axis) which...dependencies associated with each ancestry group. Since about 90% of the cell lines used in DepMap CRISPR screens...

...school. The transition has been a bit rough for her (3 weeks in and there's a big office vs. lab culture ...scientist anymore. Being a scientist is not about what you do--it’s about how you think. I have not worked at ...10 years, but when people ask me about my job, I have no qualms about saying “I’m a scientist.”    What...installment of the Addgene career guide, we’ll think about what causes this culture shock and how to overcome...counterpoints to some of the causes of culture shock above, but after polling some Addgene colleagues, we’ve...This is probably because you don't have to worry about experiments taking longer than expected or dealing... hour days at the lab, I encourage you to think about how much of that time you’re actually spending doing...

...use this ID to name the corresponding Excel sheet.3. Be specific  - When assigning a physical location...is the Laboratory Operations Manager at Addgene.  In addition to helping with daily laboratory operations...through barcoded tubes that are scanned into our Laboratory Information Management System (LIMS), a database...available for distribution!    Check Out How We Think about Developing Lab Management Software    The plasmid's...the correct sample. Tracking samples - it's all about location, location, location There are many different...of these samples have the 2D barcodes mentioned above. These barcodes identify what type of sample is ...every barcode on every tube contains information about that tube’s appropriate location. The barcode also...

...is spent trying to raise the alpha isoform of GSK-3 to the same (deserved) stature as the beta isoform...fraction (<1%) of authors have asked for advice about the scope of the policy. End of story? Not quite...the data policy was raised by researchers worried about the effort to make the underlying data easily available...making data available, but there remains concern about the extra work this may entail.  So if access to...be archived? Simplifying data access Some of the above mentioned issues could be mitigated by on-line services...him on Twitter @jwoodgett.     More Discussions About Data Sharing: PLOS Data Sharing Policy Discussion...Scholarly Kitchen) Read more Addgene blog posts about scientific sharing ...

...Strong yeast expression promoter from glyceraldehyde 3-phosphage dehydrogenase Constitutive  Very strong,...Promoter of the arabinose metabolic operon Inducible by arabinose and repressed catabolite repression in the ...Browse All Plasmids 101 Posts Learn about Mammalian Vectors Read about Reporter Gene like Luciferase and...and operators (response elements). Promoters are about 100 to 1000 base pairs long and found upstream of...this list is a great place to start, the tables above do not delve into the tissue or development-specific...

...Magnetogenetic Ion Channels. Biophysical Journal, 116(3), 454–468. https://doi.org/10.1016/j.bpj.2019.01.003...our tissues unimpeded. Andrew and Maria dreamed about the possibilities of a magnetoresponsive fluorescent...of improved response. Then, just when Andrew was about to suggest she deprioritize the project, Maria’s...with a strong commitment to exploration and collaboration. Andrew is a physicist with a background in ...potential applications.  Andrew is a frequent collaborator and is happy to give advice and support for ...interested in working on these questions or collaborating, the York Lab is eager to hear from you. Find...

...acceptable. Aims should be mechanistic, doable by 1-3 people in 1-5 years, and contribute to resolving the...a single grant. Either way, think realistically about the time and resources required to feasibly accomplish...interrupted frequently by committee members. As mentioned above, plan to give your 10 minute overview and address... list of common questions, check out this eBook about applying for faculty positions. You need to practice...program. You should be proud of it and be excited about getting the opportunity to realize your vision. ...blog posts Resources on Addgene.org Learn more about careers at Addgene Watch our career videos ...

...100th birthday, as your mom will never forgive you… #3 Honor the work day and keep it holy Keep a regular...on the Society for Laboratory Automation and Screening (SLAS) Electronic Laboratory Neighborhood.   Work...worry less about how they will handle an emergency and this will make you much more confident about their ...

...Government (2016). 2. CDC Information Pages on Antibiotics 3. WHO Antimicrobial Resistance Fact Sheet  Additional...resistant infection or not, because scientists in the laboratory will need to isolate and grow whatever bacteria...method that is frequently used in the clinical laboratory. What can we do to stop the emergence of antibiotic...CDC. Antibiotic resistance can be scary to think about, but we can all do our part to learn all we can,...drug resistance. Additionally, she is passionate about health equity, science communication, and public...

... using AAV-PHP.B vectors? See Supplementary Table 3 in Huang et al. for a list of PHP.B permissive and... an AAV rookie  Learn about some considerations when using AAVs Learn more about using AAV as a tool for...PubMed PMID: 29175632. If you want to learn more about how PHP.B and PHP.eB were engineered, check out ...Additional resources on the Addgene blog Learn more about PHP.eB, and PHP.S, a peripheral nervous system (...

...connectivity to other tools used in research; and (3) connectivity to open source data repositories. To...RSpace is integrated with most of the tools noted above, and has a modern API. Integrations with additional...into RSpace. The kind of informal conversations about research that can take place in Slack normally never...RSpace documents to external data sources (described above) further enhances the value the repository integrations...Minisymposium on Reproducibility Read all blog posts about scientific sharing Resources on Addgene.org Find...

...Orthogonal Type VI-A CRISPR-Cas Enzymes." Mol Cell. 66(3) (2017):373-83. PubMed PMID: 28475872. Find plasmids...CRISPR Featured Topic Page Learn about RNA Targeting with Cas13a Learn about CRISPR DNA Base Editing Resources...and sequences that can be edited.   As discussed above, the ability to edit RNA has multiple advantages...

...sample to a mastermix. At Addgene, we typically load 3 dilutions onto our ddPCR plate. As the titer is unknown... droplet generator, you will have to be careful about transferring your droplets from the droplet generator...Additional resources on the Addgene blog Read more about polymerase chain reaction Browse our blog posts ...collection of viral vector related protocols Learn more about the Addgene viral service ...

...darker than full vector particles.     Figure 3: Electron micrograph of AAV vector preparation shows... before being distributed to customers. Details about our production protocols, titering methods, and ...either in-house by Addgene scientists or through collaboration with viral vector manufacturing facilities, ...generated from lentiviral preparations (described above) that are subject to a concentration step prior ...

...www.businessinsider.com/how-to-get-the-most-out-of-job-fairs-2014-3 https://www.forbes.com/sites/neilpatel/2015/05/21/...or postdoc forever, and you need to be thinking about what comes next. To that end, one of the most important...personal connection; the importance of which will be elaborated on later. As for what job fairs to consider, ...the specific position he or she originally called about, you can keep in touch and reach back out when you...

...specifically to the blue triangle protein but not the other. 3) The beads are collected by centrifugation or a magnet...proteins or nucleic acids. Read on to learn more about this versatile technique.  Immunoprecipitation ...proteins in an IP are not denatured (learn more about antibodies here!) When using Protein A/G to immobilize...eluted by boiling the beads in SDS. (To learn more about Western blotting check out our blog post The Basics...

...often be seen by sinking of the tissue (see Figure 3). This method is probably the most scalable method...Generating a transgenic plant takes even longer, is more labor intensive, inefficient, and often not reproducible... region is transferred into the plant. In the laboratory we can utilize this natural process by replacing...system. The vectors mentioned in this blog post above are ready to use vectors, but with the MoClo system...

...addiction module" regulated by guanosine [corrected] 3', 5'-bispyrophosphate: a model for programmed bacterial... more about CcdB, the toxic key to efficient cloning Read all Plasmids 101 blog posts Read about knockout...with the other toxin-antitoxin systems described above, one part of the pair is more stable than the other...

... sequence complementary to the mRNA sequence. The 3’ end of  the tRNA binds the amino acid that corresponds...usage bias Read more about plasmids in our Plasmids 101 blog series Learn more about trends in synthetic... out Addgene's Molecular Biology Reference Read about genetic code expansion Browse our plasmid collection...

... the subsequent bbmerge process.     Fig. 3: How we get bbdone with our bbduk process.    ...much information as possible from depositing laboratories and associated publications, but our quality...the process. Another design choice we appreciated about Nextflow was how effortless it was for us to switch...interface. In our next post, we’ll share our story about how we used Nextflow Tower to augment our pipeline...

...immunoprecipitation (enChIP)." Scientific reports 3 (2013): 3171. PubMed PMID: 24201379. PubMed Central...specific genomic region In 2013, the Hodaka Fujii Laboratory first described a method to purify a specific...additional research conducted in the Hodaka Fujii Laboratory, please also see our interview with Dr. Fujii...

...–798. https://doi.org/10.1016/s1097-2765(02)00496-3 Kerppola TK (2006) Design and implementation of bimolecular... FP fragments and, preferably, some information about the structures of the tested PIPs. Many split FPs...neurodegeneration. He now spends his time learning and writing about science whenever he's not off the grid.   References...

...PMID: 25599551. PubMed Central PMCID: PMC4344395. 3. Lavis, Luke D., and Ronald T. Raines. "Bright ideas... the frontiers of biological imaging.  *Worried about your budget? Email [email protected] Resources on the Addgene Blog Learn About Other Protein Tags Catch Up on Your Fluorescent ...

...plasmid fingerprinting", where you cut the plasmid into 3-8 pieces such that all (or most) fragments are small...insert, but significantly off center (ideally around 1/3 of the way from one end), and also cuts in the backbone...verification, such as DNA sequencing . In the example above, digestion with enzyme RE1 will linearize the 6200bp...that you get double confirmation. In the example above, digestion with either RE3 or RE4 will give a very...

....   The Dodt and Frank Bradke laboratories later developed the 3DISCO (3-dimensional imaging of solvent...

...the membrane is cut so each lane is its own piece (3). After incubation, blocking, and washing, the membrane...protein concentration decreases).     Figure 3: Testing the antibody’s dynamic range. A) The protein...re in the right place! In this post, we’ll talk about western blot optimization and troubleshooting.   ...can be informative, so spend some time thinking about which type of gradient can help answer your particular...overexpression. Before starting a western blot, think about how you’ll interpret the data and what level of ...

...anneal to the regions upstream (5’) and downstream (3’) of the DNA segment to be amplified. When these reagents... increase the time of the denaturing. Your 5’ and 3’ primers should be designed to have similar melting...Reverse Primers: Hybridize and are complementary to the 3’ ends of the sense and anti-sense strands of the template...manufacturer’s instructions for specific instructions about extension time and temperatures. Initial Denaturation...

... host told me and wished I had been more careful. 3. Tell a story We are biologically programmed to listen...interesting content Talk about something you know a lot about and that you care about. Don’t try to present...engaging and memorable. If you read my story above about the presentation faux pas, you are more likely...the room, so it is a good idea to start thinking about public speaking early and often. Two of my jobs ...dynamite presentation?  To that end, let’s chat about giving science talks and how to make them serve ...other people give great talks We are not talking about lab meeting here. Lifeless presentation of experimental...You want to give the audience something to think about, argue with, or learn from. Some of the best talks...

...write our AMA submission to avoid rousing the trolls. 3. The AMA process was simple and well organized. You...not too worried about selling yourself and really do just want to get information about yourself and your...where users can participate in forum discussions about a wide variety of topics. Individual forums are ...where users can ask designated scientists anything about a particular topic. Given Joanne’s expertise in... is well moderated. One of our original worries about sharing science advice on Reddit was that we’d be...answering questions. r/Science has pretty strict rules about not being too sales-y in posts which falls in line...any scientists to refer to if they have questions about science careers, women in STEM, work-life negotiation...

... remember that antibodies recognize very specific 3-dimensional epitopes. Different sample processing ... Learn about research applications of antibodies in Introduction to Antibodies Learn more about fluorescence...Stay tuned for a future blog post to learn more about these techniques!  Direct vs. indirect immunofluorescence...different approaches to be familiar with when talking about IF, direct and indirect. For direct IF, researchers...immunofluorescence procedure with key considerations about each step. Image created with BioRender.com ...of protocol you will need to use. (To learn more about antibody validation, this commentary by Uhlen, et...Fluorescence Microscopy Techniques is Best for Me? Read about another common antibody application in The Basics...

...pCas into a non-temperature-sensitive replicon, and 3) added the sacB gene for counterselection to yield...complete editing process. The MUCICAT system While the above systems are great for experiments that require only...serve to interrupt genes (or gene clusters) in metabolic engineering. So far, we’ve used the MUCICAT system...when compared to the industrial strain. In our metabolic engineering case study of N-acetylglucosamine ...Multiple induction systems to choose from. Ready for metabolic engineering with improved cargo dedicated to gene...other CRISPR methods for bacteria Find all articles about microbiology Resources on Addgene.org: Visit the...

... restraining further replication.   Figure 3: Comparison of replication regulation mechanisms of... negative regulators. The genome of E. coli is about 4.6 million base pairs long and contains a single...copy number and compensating for the additional metabolic cost imposed on the host with a beneficial function.... On the other hand, they also impose a lower metabolic cost on cells and produce lower amounts of protein...

...biotechnology 16(7): 657–62. PubMed PMID: 9661200.  3. Cardin, Jessica A. et al. 2009. “Driving Fast-Spiking... on the Addgene Blog Learn More about FLEX-vectors Learn More about Cre-Lox Check out Retrograde AAVs...expression have been developed. Read on to learn more about FLP-FRT, Cre-lox, and how combinations of FLP and...

...types can be found on Addgene’s website. Fig. 3: Cas9 repressors include dCas9 alone and a dCas9-KRAB... 101 Series Learn about the PAM Requirements of Different Cas9 variants Learn about CRISPR Pooled Library...

...visualized and sorted via flow cytometry. Figure 3: Targeting pEF-BFP with a cytidine deaminase base ...for base-editing activity The reporters mentioned above all rely on GFP fluorescence and cell sorting using... Additional resources on the Addgene blog Read about other CRISPR reporters Browse our CRISPR blog posts...

...Biotechniques 35.4 (2003). 812-4. Pubmed PMID: 14579747. 3. Drexler, H.G., Uphoff, C.C. “Mycoplasma contamination...Lentiviruses are a powerful laboratory tool often employed to establish cell lines that stably express...Get Tips for Titering Your Lentivirus Learn All About Viral Vector Elements Learn How to Do Genome-Wide...

..." Gene therapy 6.7 (1999). PubMed PMID: 10455443. 3. Hindson, Benjamin J., et al. "High-throughput droplet... Addgene.org Find All Available AAV Learn More about Virus Production Find Viral Vector Protocols ...

...concentration of approximately 0.2-0.5 μg/mL (usually about 2-3 μl of lab stock solution per 100 mL gel). EtBr...different buffers and do not use water). Microwave for 1-3 min until the agarose is completely dissolved (but...cool down to about 50 °C (about when you can comfortably keep your hand on the flask), about 5 mins. Optional... of the tip of the pipette into the buffer just above the well. Very slowly and steadily, push the sample... of the way down the gel. A typical run time is about 1-1.5 hours, depending on the gel concentration ...

...Model for Regeneration and Aging. Gerontology, 66(3), 212–222. https://doi.org/10.1159/000504294 Wang,... more about model organisms in the first Five Popular Model Organisms blog post Read more about plasmids...characteristics that make them ideal for studying in a laboratory environment. We’ve covered five model organisms...have been specifically developed to thrive in laboratory settings. One of its most attractive features...minutes, they are perfect for molecular cloning and metabolic engineering.  The E. coli genome has an extensive...address many areas of microbial research like metabolism. Transport, biofilms, and much more. Because ...characterized genome, and is easily maintained in a laboratory environment. What sets Arabidopsis apart from...

...thomas-kilmann-conflict-resolution-model/, accessed Jan. 3, 2022.     Additional Resources on the Addgene blog...rely on what I know about those particular teams at that given time. I think about how previous iterations...disagreements. In fact, conflict resolution isn’t just about resolving conflicts between other people, but resolving...normally get along well with each other disagree about a specific decision, direction, or goal. Resolving...of lapsed communication. Making some assumptions about how something should be communicated is necessary...confusion around... Ownership: Misunderstandings about who is responsible for what tasks or projects is...conflicts.  Transitioning a conflict into a collaboration may not be the easiest resolution to accomplish...

...strain. F- Δ(ara-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15 λ- e14- mcrA0 relA1 rpsL150(strR) spoT1...thiamine metabolism Requires exogenous thiamine for growth ara disruption of arabinose metabolism pathway...little prokaryotic minions that carried out the labor-intensive process of replicating your new plasmid... Phage resistance gal Mutation in galactose metabolism pathway Cells cannot grow on galactose only ...repressed more   LacZ β-galactosidase activity abolished   lacY Lactose permease inactivated, lactose...plasmid stability recBCD Exonuclease V activity abolished Increased plasmid stability, reduced recombination...

...recognizes a different PAM site and generates 5’ and 3’ staggered ends after dsDNA breaks. The Doudna lab...Addgene Blog Learn about using toehold switches to detect Zika virus   Learn more about targeting RNA with...time-consuming, and complex to use outside of standard laboratories. In the case of the COVID-19 pandemic, qPCR ...with Cas13a (C2c2) Learn more about Cas12a (Cpf1) and its multiplex genome editing abilities Resources...

...Sciences, 24.9 (1999b): 359–363. PubMed PMID: 10470036. 3. Said, H. M. “Biotin: The forgotten vitamin.” American...uses of biotinylation in molecular biology! Learn about in vivo biotinylation of bacterial fusion proteins...covalently attached to the active site of key metabolic enzymes including biotin carboxylase and decarboxylases... organic acids to produce important cellular metabolites. Biotin acts as a carboxyl carrier from one compound...such as gluconeogenesis, lipogenesis, amino acid metabolism and energy transduction. Biotinylation is the...

...protein." (1998): 509-544. PubMed PMID: 9759496.  3. Buckley, Anthony M., et al. "LOV-based reporters ...deleting residues. Only about 10 amino acids can be deleted from the C-terminus and about 5 from the N-terminus...cylinder with a length of 4.2 nm and a diameter of about 2.4 nm (Hink et al., 2000). The complete beta-barrel...based on cofactor binding proteins as described above. The smallest tags are peptides that are engineered...

...base editor development going forward. Figure 3: Mechanism of gGBE. Created with BioRender.com. ...In our last post, we talked about the first base transversion editors: CGBEs, or C → G Base Editors. ...apyrimidinic sites and abbreviated as AP sites. A collaborative team from the David Liu and Dali Li labs reported... CRISPR 101: Cytosine Transversion Editors Read about four base editor reporters Resources on Addgene.org...

...BioTechniques 2015; 58:161-170. PubMed PMID: 25861928. 3. Jiang W, Zhao X, Gabrieli T, Lou C, Ebenstein Y, ...the annealed and filled-in gaps The great part about this mix of enzymes is that they can all work at...the standard Gibson assembly techniques described above, this resulted in the direct and seamless cloning...Blog Browse All of Our Plasmids 101 Posts Read About Other Cloning Techniques Additional Resources on...

...recipient plasmid to insert ratio of approximately 1:3. Since the number of base pairs for each varies, it...colonies and check them for successful ligations. Pick 3-10 colonies depending on the number of background ...Information about plasmid cloning by restriction enzyme digest (subcloning), including design and experimental...window) New England Biolabs for more information about restriction enzyme buffers). If you select enzymes...

...VWR, EX0276-1 Benzonase 250 U/µl, Millipore #71205-3 Polybrene 10 mg/mL, Millipore, TR-1003-G Molecular...mL glutaGRO 50 U/mL benzonase: 15 mL DMEM Complete 3 µL of 250 U/µL benzonase Procedure Transducing Cells...0.03887375114 6.22E+06 2 400 768 18360 0.08366013072 6.69E+06 3 200 1620 15840 0.2045454545 8.18E+06 4 100 3180 20540...the titer. Calculations In the experimental setup above, the following virus dilutions were used to transduce...v = Virus volume, mL *In the experimental setup above, the cells per well is 300,000 and the virus volume...