...errors in the design of cloning strategies set experiments back days or even weeks. Primer design was done ... alleviate these problems through good software design. But what makes software good? Fortunately, that...we dug into the original literature, some of it decades old, to provide reliable feature annotations. Even...

...resources to provide them to their students. My desire to create a set of low-cost, easy-to-use educational...protein – you first need the DNA sequence that encodes it. That DNA sequence then needs to be incorporated...one is as cheap as 10 cents, making activities designed with FD-CF reactions affordable to many schools...students. In our BioBits kit for example, we’ve designed FD-CF experiments that result in outputs that ...

...emission at 560nm. Shortly after the initial article describing the cloning of firefly luciferase was published...the plasmid, which is then introduced into the desired mammalian, plant or bacterial cells. Expression...stop codon, which is then transfected into the desired cells. A hybrid mRNA transcript containing luciferase...expression and more complex cell treatments can be designed to further explore the pathway. Which luciferase...

...but can be minimized with high powered research designs. Unanticipated discoveries Finally, there are good...rates, should be reported as a new discovery that deserves confirmation with new data and a new, preregistered...Guidelines; preregistration to increase clarity in study design and analysis; removing publication bias with Registered...Science, Dr. Mellor worked with citizen scientists to design and implement authentic scientific research and...

...purification is now a reality thanks to OptoNBs. Recently designed by the Toettcher Lab, OptoNBs are photoswitchable... put AsLOV2 because here it was less likely to destroy the nanobody’s function than if inserted into the...blue-light induced binding. The lab used this general design scheme to generate two more OptoNBs, one against...exciting because using light to control signaling provides greater spatiotemporal resolution than pharmacological...

...Addgene provides the scientific community? Katz: The most important resource that Addgene provides is an...is continuing to look at providing other things besides plasmids to the scientific community through Addgene...exciting about the open science movement is that it provides interesting and unique challenges for intellectual...

...’s social media toolbox. Besides advice for using Twitter, this post provides suggestions for using LinkedIn...use Twitter as a scientist. While this post is designed with beginners in mind, there’s some great reminders...sheet from Science Media Savvy This tip sheet provides advice for tweeting and blogging as a scientist...

...target specific mRNA transcripts for degradation. Despite how easy these experiments are to physically set...to prevent the incorporation of non-specific nucleotides into the dsRNA leading to increased RNAi efficiency... Lab’s recent Nature Biotechnology paper, they describe TurboID and miniTurbo, which utilize non-toxic...switch to be expressed. In this case, the gene encodes the LacZ𝛼 protein. Once expressed, LacZ𝛼 complexes...

...trillion connections (synapses.)  In this post we’ll describe the AAV retrograde serotype (AAVrg) and how it... this can cause challenges for the experimental design as these vectors can exhibit spurious expression...efficiency can have a major impact on experimental design and interpretation of results. Take extra care ...References 1. Tervo, D. Gowanlock R., et al. "A designer AAV variant permits efficient retrograde access...

...New work from Konermann et al. and Yan et al. describes new Cas13d enzymes that average only 2.8 kb in...Like other Cas13 enzymes, the Cas13d orthologs described in these papers can independently process their... high targeting efficiency, both of which are described below. Yan et al.: WYL-domain-containing accessory...produced very similar cleavage products even when guides were tiled across a target RNA, indicating that...

...Harvard University. In my previous two posts, I’ve described the fundamentals of how to work with Drosophila...Drosophila as an experimental model organism. I then described the Gal4/UAS system used by geneticists to study....e. there won’t be positioning effects). When designing experiments where you want to express multiple...wondering, “How do I know which of my flies have the desired integration?” Not to worry! In flies, phenotypic...

... NEB's Resource describing Dam and Dcm Methylases Discovery of DpnI Review describing dam-/dcm- E. coli...mutagenesis. During this process, incorporation of a desired mutation into your plasmid of interest by PCR generates...only the non-mutated and methylated template is destroyed leaving behind a pool of mutated plasmids which...

...cleaved precisely as predicted with no addition of nucleotides to the 5’ spacers. The 3’ ends were modified ...advantage over other methods that favor certain nucleotides at the 5’ end. PTG construct schematics. Scissors...the plasmids made by Xie et. al are specifically designed for plant expression, PTGs will likely be adaptable...worries, I've summarized the three strategies described in this post in the graphic below - including ...

... for over 10 years. Many successful scientists describe having a “posse” of mentors as one key to their...Ellen Daniell. In “Every Other Thursday,” Daniell describes a group of 7 women scientists who met every other...increases the diversity of input and perspectives Provides a good mechanism for accountability that makes...leadership and communication skills. The leader also provides materials such as pre-reads, videos or exercises...

...shown in the figure below. Their most successful design utilized Magnet photoswitchable proteins derived...architecture. Yu et al., 2020 FAST BphS from R. sphaeroides Split Cas9 Genome editing by NHEJ and HDR. Can...Cas9 Richter et al., 2016 tsRC9 RsLOV from R. sphaeroides Cas9, dCas9 Gene editing Zhou et al., 2018 ...al., 2016 CRISPR-plus Photocleavable ssDNA oligonucleotides Cas9, photocaged-gRNA Gene editing Manna...NPPOC-caged trimethoprim (TMP) Cas9 fused to destabilizing domains (Cas9-DHFR) Gene editing, transcription...

...in science – the time spent by the scientists designing and performing the experiments. This cost varies...quickly with the postdoc who’s really good at primer design – but first, you have to know what resources exist...well) plate! How much do repositories help?   Designing precise calculations for any of the numbers in...engagement with and use of repositories like Addgene provides large-scale savings. How large? Using Doench’s...

...and SMASh degron tagging vector! Destabilizing domains Destabilizing domains (DD) are akin to degron ...   Fig. 2 Tetracycline on/off promoters and modes of action Check out Addgene's Lentiviral Tet-on ...post-translational level, unlike most of the other mechanisms described. Fig. 3 dTag mechanism of protein degradation...

...we would be remiss not to at least mention. Oligonucleotides allow for sensitive detection of low abundance...strong protein-protein interactions with other peptides, like an Fc-binding domain (Figure 1).  ...Strong protein-protein interactions with other peptides. Created with BioRender.com.     Many biologists... conjugation. But hopefully, this introduction provides a good base from which to dive deeper!References...

...molecular biology experiments was in the job description, but this skill is not just about reacting to...puzzling through your broader research questions and designing future experiments. Being able to work through...research non-academic careers, I always felt that descriptions of transferable skills seemed fuzzy or ill-defined...most likely cause, you implement an experiment designed to fix the problem. Then you assess whether your...

... plasmid maintenance during cell division. ccdB codes for the toxic protein (CcdB) that acts as a DNA ...death. ccdA, another gene found in the ccd operon, codes for the antitoxin protein (CcdA) that protects the...of ccdB, leading to positive identification of desired clones.   ...Becomes a powerful cloning tool... efficiently, as those that do not contain the desired insert should not grow.   Figure 2: Image...

... as the OG (as the kids say) affinity reagents deserved to be celebrated, there are many other amazing...its thorns and every affinity reagent has its downsides. These reduced-in-size options often have reduced...°C and can refold correctly after denaturing.  Designed ankyrin repeat proteins, or DARPins, are genetically...a wide variety of targets, including proteins, peptides, amino acids, drugs, metal ions, and cells. They...

...another. If you are using barcodes (and you should be!) bear in mind that your barcodes are being applied to...containers, not to the contents. Tightly coupling barcodes and containers might not be such a bad idea, tightly...Resources on the Addgene Blog Learn about How We Use Barcodes in the Lab Get Tips on How to Keep a Lab Notebook...

... nuclease activity of the Cas9 nuclease itself. Design and testing of eSpCas9 Looking at the structure...and the negatively charged DNA and therefore destabilize basal strand separation. This decreased strand... choice and get improved on-target specificity. Design and testing of SpCas9-HF1 Kleinstiver et al and...Joining Repairs Double Strand Breaks Get Tips on Designing Your gRNA Browse Our CRISPR 101 Series Resources...

...tools, they do have undesired off-target editing effects. ABEs have been described to introduce base changes...efficient. Informed by these mutations and previously described TadA mutations (Gaudelli et al., 2017), they modified...heterodimeric fusion of wild-type TadA and TadA7.10, they designed one set of ABE8’s similarly (ABE8.x-d). In order...

... allele decreases. Crossing mice to generate a desired phenotype Instead, genetic engineering should be...in the journey towards producing mice with your desired combination of mutations. Assuming your parental...generate mice that are homozygous for all of your desired genes. Remember Punnett squares from high school... are homozygous (or hemizygous) at all of your desired alleles to conduct your experiments. You should...

... They fused the Cas enzyme to cell-penetrating peptides and co-incubate it with an “assist peptide” that...with a 30-minute incubation, even with multiplexed guides. Figure 1: A) Cell-penetrating Cas9, fused...all applications. Addgene’s plasmid collection includes over 10,000 plasmids with a FLAG tag (!), but ...recombinant antibodies as well? Our collection includes a mouse and a chimeric rabbit (original mouse ...

... developed based on 100 structure-guided spike designs and has improved protein yields and stability. ...Plasmids deposited from Alejandro Balazs’s lab includes those for generating pseudovirus and those encoding...Liu Y, Chou C-W, Byrne PO, Hjorth CK, Johnson NV, Ludes-Meyers J, Nguyen AW, Park J, Wang N, Amengor D, ...Finkelstein IJ, McLellan JS (2020) Structure-based design of prefusion-stabilized SARS-CoV-2 spikes. Science...

...newer generation lentiviral packaging systems were designed to prevent the formation of replication competent...use 3 plasmids; an envelope plasmid that usually encodes VSV-G, a packaging plasmid containing structural...transfer plasmids with small inserts (such as CRISPR guides) while a single 72h harvest is best for larger ...the best titer possible. While this information provides a great starting point, it may not translate to...

...soCoChR-expressing system. Taken together, Shemesh et. al. describe a novel system for the precise mapping of neuronal...the single-cell level. Various soCoChR plasmids described in this work including the soCoChR-GFP plasmid...information about spatial and temporal organization. The design use to detect PKA activity can be generalized to...choice for FRET assays where a dark acceptor is desired due to its very low quantum efficiency. ShadowY...

...for affecting change. On Friday, the symposium includes a panel discussion and keynote by former congressman...towards solutions. The 2015 Boston FoR meeting was designed to explore areas where further data is necessary...for students interested in leading the process despite the recent advance. Similarly, grad students throughout...development and implementation of new strategies designed to cope with current increases. These two rulings...

...for Cas9 (42 nt vs 100 nt.) However, the newly described non-CRISPR protein NgAgo is smaller than any of... of these options (2.7 kb with 24 nt guides), so it has the potential to eclipse any of the CRISPR nucleases...to examine Cpf1 off-target editing. First, they designed gRNAs with single and double mismatches throughout... endjoining (NHEJ). Cas9-mediated NHEJ usually destroys the PAM site due its proximity to the cleavage...

...switches? FLEx (or “flip-excision”) switches were designed as a genetic tool for researchers to conditionally...does the FLEx switch work? Let’s say you want to design a genetic FLEx switch that turns BFP expression...single gene deleted in your knockout mouse, you can design a FLEx switch conditional knock-in cassette. As...experiment with a wildtype copy of a gene, you can also design FLEx switches to conditionally test the function...

... thanks to our guest blogger, Laura Desrochers! Laura Desrochers is currently a Postdoctoral Scientist...Apr 6, 2021. This post was contributed by Laura Desrochers, a postdoctoral scientist at AstraZeneca. Why...ve discovered.) As someone who has now seen both sides of the postdoc experience, I’m going to share what...

...navigate a global pandemic can also be the thing that provides a new perspective that helps to clarify the harder...to introduce yourself to a few members of your desired department during the application process. Many...However, an online meeting with members of your desired program will allow for you to have the time and...during my application cycle, two schools on opposite sides of the country had scheduled visitation days during...

...interest from its production site to its final destination. As an example, most secreted proteins are produced...pathway to their subcellular or extracellular destination. At the end of their life cycle, proteins may... about the subcellular structures your protein resides in and potentially which other proteins it forms...in Figure 2 (adapted from 4) - Dunn et al. 2011 provides an in-depth review of quantitative colocalization...

...results can be affected by DNA quality and primer design, particularly for amplicons under 100 bp (Ma, 2015... concern, look for the lowest-cost option that provides you confidence in the results you need.  Whichever...by running the product on a gel. And only ddPCR provides absolute quantitative data, instead of relative...Polymerase Chain Reaction (PCR) Protocol: How to Design a primer  Molecular Biology Reference Molecular...

...die from cancer-related complications each year. Despite these grim statistics, new strategies for attacking...cancers, especially those of the pancreas and colon. Despite the prevalence of Ras mutations in a variety of... enabled researchers to begin discovering and designing molecules that are showing some promise in their...in the screenshot below). Currently, the page includes interactive maps for the Ras, mTor, and p53 pathways...

...researchers must co-transfect cells with 5’-P-ssDNA guides and an NgAgo expression plasmid to edit a gene ...Try NgAgo for Yourself On the other hand, the widespread success of CRISPR-Cas9 across multiple model ...vector or in vitro synthesis, which could be less desirable if one has to target multiple sites in a single...of the supposedly alien 5’-phosphorylated ssDNA guides, which might be rapidly degraded by the cellular...

...with various spacer lengths from 30-84 nucleotides, and designated this system REPAIRv1. Using next-generation...introduced through non-homologous end joining (NHEJ). Designing an RNA editor The Zhang lab envisioned a two-component...that interact with duplex RNA, they hoped to destabilize ADAR-RNA binding to decrease off-targets. Of ...

...are no exception to this effect -- meticulously designed experiments in labs around the world were put ...requests correlate with paper publication The dataset includes 11,426 plasmid requests over 6 years for 35 papers...into two components: 𝜃x(t) and 𝜓z(t). 𝜃x(t) describes the exponential decay process we observed, while...model for each region using stochastic gradient descent in Pytorch, which gave us the maximum likelihood...