...obstacles that need to be overcome. Conflict, and its inherent tension, is interesting. It’s what drives a story...actually from a completely unexpected finding in a different experiment, this tool was developed because a ...couldn’t progress without it… These details often aren’t included in scientific communications, but they...scientists doing the work.  And while these pieces aren't always appropriate for your formal scientific communications...doing and sharing science in the first place?   References and Resources More resources on the Addgene blog...

...due to genetic drift and the use of genes from different microbial strains. It proved to be impractical... least 96% sequence identity when detecting a reference feature. For a coding sequence feature that may... could miss a common feature if the sequence differences exceed the threshold. This issue is addressed...often come in closely related versions that have different properties. To prevent misidentification, we augment...way for SnapGene users to apply features from a reference strain to newly sequenced strains through our ...

...cells. The replicons of plasmids are generally different from the those used to replicate the host's chromosomal...include ColE1, pMB1 (which comes in a few slightly different but well known derivatives), pSC101, R6K, and ...considerations. *Requires pir gene for replication (reference). **F1 is a phage-derived ori that allows for...chloramphenicol – make sure your growth conditions aren't working against you!  The culture inoculum ...clarified? If so please, post in the comments.  References Chatterjee A, Sun SB, Furman JL, Xiao H, Schultz...

...AAV. You’ve never worked with AAV before, but you aren’t going to let that stop you. Where do you start?...capacity between the ITRs is ~4.4 kb. ITRs are inherently unstable due to their secondary structure, palindromic...species or location. Promoters can also drive different levels of expression. For example, CAG is a strong...Next let’s talk about some of the elements that aren’t required parts of an AAV transfer plasmid, but ...specific expression of your gene of interest. References Haery L, Deverman BE, Matho KS, Cetin A, Woodard...

...antibodies for tagging purposes. Different antibodies bind different protein sequences called epitopes...infamous frankenstein, was constructed by grafting different parts together. By combining two optimized elements...living cells. To make these scaffolds specific to different targets, the scientists fused them to sequences...to build a more robust set of frankenbodies. References Ewert, S., Honegger, A., & Pluckthun, A. (2004...

... our values of transparency, reproducibility, and community-building. The transparency begins with sharing...whether any given antibody is available through different companies (Bandrowski et al., 2016). It’s also... the antibody sharing landscape as well.     References Andrews NP, Boeckman JX, Manning CF, Nguyen JT... Improving Reproducibility through Rigor and Transparency of Experimental Methods. Neuron 90(3):434-6....

...previously hidden epitope (right). Created withBioRender.com.   Biological Processes  If you’re feeling...outside of the cell. The same protein may have different epitopes available if it is being selected out...controls in validation, as you may need to test different configurations and conditions to understand exactly... what a given protein is doing or changing in different conditions. Just remember to keep good records...when you’re building your antibody library! References and Resources More resources on Addgene’s blog...

...Argonaute proteins play an important role in RNA interference, binding RNA guides to cleave foreign RNA. Argonaute...guides/gene for 10 genes, they did not observe differences in cleavage efficiency. If this finding is replicated...) or staggered (Cpf1) cleavage. NgAgo takes a different approach, randomly removing 1-20 nucleotides from...Read About NgAgo User's First Impressions Here References 1. Gao, Feng, Xiao Z. Shen, Feng Jiang, Yongqiang... 2. Swarts, Daan C., et al.  “DNA-guided DNA interference by a prokaryotic Argonaute.” Nature. 507(7491...

...transition from a clump of cells to a fully differentiated body. In contrast, these dynamic processes ...distinguishing cell populations that have been tagged with different fluorescent proteins or dyes. 3. Two-photon microscopy...cells. For example, by harvesting lymph nodes at different times after antigen challenge, scientists were... see movement and interaction between several different immune cells. These methods allowed immunologists... like this is a mosaic of cells labelled with different fluorescent proteins. Using one iteration of this...understanding of the immune system and its dynamics. References 1. Peters, Nathan C., et al. "In vivo imaging...

...isotype is expressed at different times of the immune response and initiates different immune cascades. In...In the research setting, antibodies of different isotypes can be used together in the same experiment ...However, each animal will make antibodies against different protein epitopes and so variability between lots...types of antibodies are out there, and how many different research applications they can be used for. Addgene... - and we’re always growing our collection! References: Arbabi-Ghahroudi M (2017) Camelid Single-Domain...

...globe, it can be a bit of a mystery for those who aren’t familiar with it. If you’re curious about the hows...is shared between two parties. There are many different flavors of MTAs, but the Uniform Biological Material...which sometimes makes people think our MTAs are different than other institutes. But they’re not! The vast...perfect, but it does provide consistency and transparency in an otherwise messy process.  And you do have..., instead of coordinating signatures from two different institutes. Our online portal automates a lot ...

...have risen into yeast expertise! References and Resources References Liu, Wei, et al., From Saccharomyces...lists some of the biological commonalities and differences between K. phaffii and other yeast strains.   ...Applications for K. phaffii There’s a number of different uses for the phaffii-lous strain. Protein expression...The yeast giants - S. cerevisiae and S. pombe - aren’t going anywhere anytime soon, and for good reason...

...that replicate current knowledge, but the sensor can also kinetically outperform current methods. Unger...intended to help early career scientists attend a conference of their choice to share their fluorescent protein...are then extracted and sequenced. Chiasson is currently using VAMP-seq to study vitamin K epoxide reductase...and rapidly test the variants in vitro against different drugs and dosages.  Michal Bernach: Developing...

...for creative interaction During Zoom meetings, different tools can be used to promote creative interaction...as a picture. I believe that showing something different from time to time breaks up the monologue a little...papers will be written by several authors from different institutes, disciplines, and cultural backgrounds...and only general chatter will be allowed with a different host each time that moderates conversations. This...communities. You can follow him on twitter @salmon_phd References Frassl MA, Hamilton DP, Denfeld BA, de Eyto E...

... the library are differently sized, smaller plasmids may be amplified preferentially, skewing the composition...successful amplification? To provide a point of reference, Addgene commonly prepares NGS samples using both...overrepresented plasmids (plasmids that were amplified preferentially). You can also rank the plasmids by abundance...create a “cumulative wealth distribution” curve, or Lorenz curve. By calculating the area under the curve ...

...based on the predicted sizes and organization of different features within the plasmid. Restriction analysis...can search NCBI for YGOI to find the particular reference sequence if necessary. The example plasmid on ...fragment having increased intensity to serve as a reference band. The uncut DNA (lane 2) shows 3 possible ...in the gel well and the dyes provide a visual reference point so you can easily assess how far the gel...Eductional Resources: Addgene's Molecular Biology Reference Find More Protocols and Videos      ...

...far the T4 exonuclease proceeds: at the first occurrence of the chosen nucleotide, T4 will switch to polymerizing...efficiently repair the plasmid. Created with BioRender.com.   SLIC also opened up a new world of multicomponent...bacteria, which repair the plasmid. Created with BioRender.com. How does SLIC compare to other cloning ...method’s flexibility allows it to be used with different types of PCR-generated inserts. By harnessing ...updated by Emily P. Bentley in September 2024.  References and Resources Further reading Harnessing homologous...

...organizers. My overall impression from this conference was that Cancer Researchers and Immunologists...Here I aim to compile a few highlights from the conference which reflect how cancer/immunology researchers...multi-pronged attack. Inflammation and tumorigenesis The conference opened with a very interesting presentation by...our Fluorescent Protein Guide. Throughout the conference aspirin and the use of COX inhibitors was discussed...about our free deposit and distribution service! References: 1. Pietrocola, Federico, et al. "Caloric restriction...

...tools to further their research. However, tools aren’t very useful if you don’t know how to use them. ... ideas come from social media conversations, conferences, and questions our scientific support staff receives...line with Addgene style.        Fig. 2: Reference image (left) provided to our graphic designer ...for our blog posts, we usually make figures in BioRender or use appropriately licensed and cited figures... We are constantly reviewing the resources we currently offer to identify gaps and add to them as techniques...

...Sanders of ComSciCon, the Communicating Science Conference series for graduate students. I believe that...giving a public talk, ask a friend who works in a different field to listen to you practice.  Ask them what...organizations. At ComSciCon, our primary mission is to strengthen and support the network of graduate students ...addition to this annual event, graduate students in different regional and disciplinary communities gather at...Learn about careers in science communication The strength of story telling Science communication: 9 strategies...

...chromosomal domains, or how stretches of DNA on different chromosomes interact with each other. Chromatin...Architecture and Chromosome Organization. These aren’t the only investigators in the chromatin field who... Note that histone genes frequently have many different variant names, not all of which can be listed ...Hutchinson JN, Sara SA, Ensminger AW, Fox AH, Chess A, Lawrence JB Mol Cell. 2009 Mar 27;33(6):717-26. Reporter-nanobody...me at the Keystone Chromatin meeting, especially Karen Reddy (@theReddyLab), Thoru Pederson, Joanna Wysocka...

...developers work in a streamlined manner. Now, in my current role at Oxford Nanopore Technologies, I work with... part of my job is getting to talk to so many different people! I communicate with customers, software...Often, product managers will have three or more concurrent projects in varying stages, which can get messy...meetings. Meet with my boss and coworkers, discuss current and future features being built for the analysis...blogger Rachel Rubinstein! Rachel Rubinstein is currently a Field-based Product Manager at Oxford Nanopore...

... a screening strategy as well. In a screen, you aren’t killing off a portion of the cells like in a selection...You’ll want to pick enzymes that will give you different sized bands depending on whether the plasmid contains...using vector-specific primers, look for size differences. You can also use primers where one primer anneals...popular sequencing primers in our Molecular Biology Reference. Sometimes common  primers can't provide all of... Sanger sequencing Read the Molecular Biology Reference ...

... scientific conference, whether you are attending in person or monitoring that conference via social media...American Society for Cell Biology (ASCB) 2018 Conference. I don’t exactly know how I was selected to participate...like Fast Company, opportunities to speak at conferences, and offers for postdoc positions. Check out ...s worth a read since many of the pointers are different from the other links shared in the post. And the...

...commonly used in research laboratories to detect the different subclasses, but there are also secondary antibodies...secondary antibodies to detect multiple targets with different mAbs in the same experiment. The Trimmer Lab found...within the plasmid backbone to generate R-mAbs of different subclasses, including subclasses that were not... useful research materials and information. References and Resources Additional Resources on the Addgene...Addgene's recombinant antibody plasmid collection References Andrews NP, Boeckman JX, Manning CF, Nguyen JT...

...the wrong everything else.      References and Resources References Boston University Flow Core. Fluorochromes...standard curve, another quick experiment, or using a different vial and saving the poor, abandoned antibody for...assays involve multiple antibodies, each with a different signaling molecule, that all need to be detected...   Fig 2: Try to inversely pair fluorophore strength with protein abundance.    Second, you’ll ...

...Linearized Plasmids After PCR). Current site directed mutagenesis methods Current SDM methods rely on in vitro...those to PCR amplify the template plasmid. The parental, nonmutated DNA is digested with methylated DNA-specific...common methods for site directed mutagenesis   References 1. Trehan, Ashutosh, et al. (2016). “REPLACR-... on Addgene.org Browse Our Molecular Biology Reference Pages Choose Your Perfect Plasmid Backbone Find...

...journal.pgen.1004086 Ghezraoui H, Piganeau M, Renouf B, Renaud J-B, Sallmyr A, Ruis B, Oh S, Tomkinson AE...repair are easily re-cleaved while indel products aren’t, so repeated cycles will favor accumulation of ...Comprehensive Cancer Center at UNC Chapel Hill.   References Bennardo N, Gunn A, Cheng A, Hasty P, Stark JM...2014) Is Non-Homologous End-Joining Really an Inherently Error-Prone Process? PLoS Genet 10:e1004086 ....

...dependence on excitation power is important since different fluorescence imaging strategies use widely varying..., TIRF or spinning disk confocal use entirely different excitation power regimes. Therefore, it is unpredictable...the best way to compare the photostability of different fluorescent proteins? Measuring photostability...excitation power. By repeating these measurements for different fluorescent proteins and by comparing how the ...measurements reported in the literature are performed in different ways. We describe a number of issues with some...

...protein expression journey! Resources and References References Nabet et al. The dTAG system for immediate...expression of essential proteins (when knockouts aren’t viable). siRNA or RNAi are small, double stranded...days. There’s also a CRISPR for that! CRISPR interference (CRISPRi) targets a catalytically dead Cas9 ..., and expression is tunable by titration with different amounts of tetracycline. Tetracycline regulation...