...University of Amsterdam. Before you decide which car you want to buy, it is worthwhile to test-drive a couple ...the best advice is to try a couple of promising variants to check how they perform under your experimental...protein properties need to be scrutinized under relevant conditions. The properties of fluorescent proteins...detected signal and therefore high brightness is an important feature of a fluorophore. The key question is ...indicated by the extinction coefficient (EC) and quantum yield (QY) respectively. The theoretical brightness...However, the theoretical brightness ignores several important experimental conditions that are related to the...fluorescent protein in the host cell, which is a key determinant of the practical brightness. Therefore, practical...

...Mentoring Group all participants act as both mentors and mentees. Because the participants are at a similar...make mentoring less mysterious and train the participants in skills useful for future mentoring relationships...step-by-step, how-to guide to get one started. Advantages of the group mentoring format Adding peer advice...at the same time Broadens the network of the participants Can be a lot of fun! Form your own peer mentoring...talk to trainees more about peer mentoring. They want to find mentors to help them develop their skills...Discuss your general areas of interest and what you want to cover during the year. Suggest that each person...perhaps a 3 hour breakfast once a month. All participants must commit to making the meetings a scheduling...

...reasons why doing something without a kit has advantages: less plastic waste, less expense, and less of...such as RNaseZAP. When harvesting tissues, cells, plants, fungi, or bacteria, keep samples cold and work...effectively isolate intact RNA, a fast, strong protein denaturant would be required- something that broke down ...Chirgwin and colleagues showed that a strong protein denaturant, guanidinium thiocyanate, did just this (Chirgwin...Chirgwin et al., 1979). They developed a protocol meant for isolating RNA from rat spleens in which they...material is removed via centrifugation and the supernatant is extracted with chloroform as in the above ... results of a downstream application such as quantitative PCR to assess gene expression. There are several...

...al. were interested in correcting an autosomal dominant mutation in MYBPC3 that leads to hypertrophic ...in cases of disease prevention. However, it is important to consider that already-tested CRISPR alternatives...cardiomyopathy and other genetic disorders, pre-implantation genetic diagnosis (PGD) screens embryos to find... find those that do not carry a given dominant disease-causing gene, or that carry only one copy of a ...clinical applications could be considered. It is important to consider the question of broader applicability...had few predicted off-target effects, but this advantage likely won’t transfer to all genes we’d like to... large step forward in the CRISPR field, it’s important to remember that the study hasn’t achieved any...

...from countless rounds of optimization. Dozens of variants exist with unique cutting features and evolved...engineering endeavors. For more information on Cas9 variants, check out our blog post on choosing a Cas9 enzyme...effective at wiping out large regions of DNA and has anti-viral application potential as a result. Fast facts...enzymes outlined below as well. Cas12a also has significant therapeutic promise due to the fact that it is...target sequence Similar to Cas12, it is also significantly smaller in size than traditional Cas9. Another...when engaged by its target sequence. Why would you want to give it a go? The high specificity of Cas14 makes... detection tool for rare, often hard to detect variants such as single nucleotide polymorphisms. Fast...

...for oxidizing environments to photoconvertible variants, it seems like GFP is always learning new things...frustration many researchers have experienced: wanting to ask and answer experimental questions, but not...needed to do so. Cepko lab researcher Jonathan Tang wanted to express a variety of proteins in single cell...nanobodies, the project really took off. Unlike most antibodies, nanobodies, which occur naturally in camels,...optogenetics in specific cell populations even easier. Advantages and other possibilities It’s clear that Cre-DOG...GFP-labeled lines, and they each have their own advantages. T-DOGGs can be made drug-inducible using the...proteins with a modular structure, constructing split variants should be relatively straightforward, but it is...

... controlled by regulatory networks in the plant. If we want to access that type of material - or do what... therapeutics, if you want to program a cell to identify and target a malignant cell, then you’d need ...talk about off-target interactions. That’s relevant if you want to make sure you don’t target anywhere else... parts are required to get the function that you want. The last few years, we’ve really been making tons... sophisticated. There are also proteins, like antibodies. But we are really just using what biology has...

...fusion protein that switches to any color or tag you want? It sounds like magic, but it’s real! In this post...photophysical properties of chemical dyes provide many advantages over fluorescent proteins, opening up new applications...HaloTag is actually HaloTag7, and recent specialized variants run up to HaloTag11. HaloTag is now the most popular...for every new-and-improved fluorescent protein you want to try, just swap in a new fluorescent ligand and...decorate synthetic viral particles with BG-linked antibodies to drive cell type-specific delivery of a viral...any cloning at all! As mentioned above, several variants of these tags have been developed over the years...are typically 230–250 aa. Smaller size isn’t a guarantee that a tag won’t disrupt the function of the target...

...set out to make my first CRISPR/Cas9 gene edit. I wanted to inactivate the gene BRAF (a kinase implicated...expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9 Western blotting was performed on whole-cell...at the indicated multiplicity of infection (MOI). Anti-vinculin Western blotting is a loading control. ... additional 3 weeks under blasticidin selection. Anti-Cas9 Western blotting was performed on whole-cell...having active Cas9 (see tip 2). Pro-Tips 1. To quantify Cas9 activity using this assay, you really need...of the gene with multiple gRNAs, but the most important is that you could avoid potential off target artifacts...be easier to visualize and identify). If you need/want to put the digestion site in the middle of the PCR...

... can return multiple answers. You might want to take advantage of their save and recall functions when... with significant lot-to-lot variation. Additionally, this assembly, which used only antibiotic screening...Langhorst, B. W., Bilotti, K., Pryor, J. M., Cantor, E. J., Canton, B., Knight, T. F., Thomas C. Evans, J.,...you get results you like. NEBridge SplitSet If you want to design an overhang set specific to your DNA sequence...J. M., Potapov, V., Kucera, R. B., Bilotti, K., Cantor, E. J., & Lohman, G. J. S. (2020). Enabling one-pot...

... While antibiotic resistance-mediated selection is indispensable for cloning work, antibiotics and plasmid-encoded...the Addgene Blog Plasmids 101: Antibiotic Resistance Genes Antibiotic Resistance: An Old Solution but...now used routinely in biotechnology to transform plant cells. Degradative plasmids: microbial hardiness...environments — like by living where no one else wants to. Some organisms manage this with plasmids that...created as whole-genome sequencing becomes more relevant (Garcillán-Barcia et al., 2023). In this post,... antibiotic...

... produce 3 concurrent base edits to withstand antibiotic selection. As a result, 8 mutations in TadA could...modified ABE7.10 accordingly which resulted in variants named “ABE8.x”: “8” stands for the eighth round...x-d.  This resulted altogether in 40 new ABE8 variants. When comparing on-target DNA editing efficiency...replaced S. pyogenes Cas9 in the ABE complex with PAM-variant Cas9 proteins: the S. pyogenes NG-Cas9 (PAM: NG...PAM: NNGRRT) to create Sa-ABE8.x-m/d. With these variants, ABE8 could target many additional locations in...cells using 8 selected ABE8s and ABE7.10. They quantified genomic editing by measuring the reduction in... guide-independent off-target deamination, an important attribute for therapeutic application. ABE8s demonstrate...

...companies that produce GMO food plants. When a press piece ran on the detector plants, we expected to see that... lab focused on product development: building a plant that could sense and respond to novel substances...Facebook comment.  In our day job, we worked on plants engineered to detect explosives and rapidly indicate... a beautiful GMO, one that people would actually want to have. Once we had the idea, we knew it was a ...It has also allowed us to really analyze what we want out of our careers: to build something new and beautiful...we are building this company, and that’s what we want it to do. It’s exhilarating to have that sort of...have great partners in this accelerator program who want us to succeed. We’re excited to see what the next...

...third post will describe how you can make your own mutant flies. Find Drosophila Resources at Addgene The...combs. To make genetic crosses properly, it is important to isolate females from males while they are still... after hatching into their adult phase. It is important to clear out the parent crosses from the container...method for maintaining stable lines of genetic mutants, including in situations where maintaining heterozygosity...feature of balancer chromosomes is that they produce dominant phenotypes visible on the flies. For example, ...feature when setting up genetic crosses. More importantly, this knowledge will stop you from embarking ... of Fly Pushing by Ralph J. Greenspan. It’s a fantastic book for newbies on how to work with flies. ...

... and this nuclease-null or “dead” Cas9 (dCas9) variant can then be fused to effector domains such as transcriptional...limitations to a system that relies on two distinct Cas9 variants—nuclease-competent or nuclease-null—in order to...with both the nuclease-positive and nuclease-null variants of Cas9 along with the necessary gRNAs, but it...control which gRNA becomes complexed with which variant of Cas9. To circumvent this problem, previous studies...suffers from several limitations. Perhaps most importantly, most Cas9 orthologs are less well characterized...target site. Benefits of truncated gRNAs Importantly, the use of truncated guides has largely been...graduate student at the University of California, Santa Cruz, where her research is focused on engineering...

...strains, a change in media may be advantageous. Reduce the antibiotic concentration Low copy plasmids produce...additional steps are needed to optimize yield. You might want to include an extra wash step or use a different...suggest retransforming the plasmid into a strain meant for DNA purification. It sounds like extra work,...many high copy plasmids, standard LB broth and antibiotic concentrations will work just fine; however, ... produce fewer transcripts of their antibiotic resistance genes and can therefore be cultured in media...media containing half the normally recommended antibiotic concentration. Add supplements to the media You... Resources on Addgene.org Find Info on How to Quantify Your DNA Learn to Do a Bacterial Transformation...

...differently and you may want to select one (or both) depending on the information you want to verify. We will...Organism box. This small piece of information can significantly reduce your wait time for blastn, blastp, and...expected ORFs, mutations or truncations. A primary advantage of blastx is that you do not have to decide on...using blastx at Addgene, we use the default “Non-redundant protein sequences (nr)” database as it contains...recommend using blastp over blastx. The primary advantage is time savings but an added benefit is that blastp...the default scoring matrix BLOSUM62, but you may want to check the description of the other matrices to... to see if another would be more advantageous for your search. Timesaving Tip #3: Note that protein databases...

...for science, and numerous other reasons. It is important to remember that we, scientists and non-scientists...role of science in our democracies will require constant vigilance and actions beyond marches, as well ...and calling for billions of dollars in cuts to important research funding.  So when I saw that there was...depends on it. Deanna McLafferty is an editorial assistant and activist. Washington, DC, USA 38.9072°N 77.0369...scientist.  I care about my work because it's important to our health.  Most people won't argue with that...been so well-publicized, we might encounter hostile anti-science activists. However, I must say that I was...I’ve worked tirelessly to get the publications, grant funding, and teaching experience that it takes to...

...CRISPR in plants, to learn techniques for detecting genome edits, to discover why you might want to use ... sent Outreach Scientists to Colorado, Berlin, Atlanta, Ottawa, Norwich, and Hannover, we raised money...popular blog post this month shares incredibly important information for anyone considering a postdoc. ... through your PhDs. Of course there were many fantastic posts in October. Click on the links below to ...Advice Part 2: Building Community Engineering the Plant Genome Using CRISPR/Cas9 Healthcare Consulting: ...Door to the Business of Life Sciences 6 Tips for Grant Writing 5 Reasons to Use Reddit for Science Communication...

...in close proximity and present in large enough quantities (and the correct stoichiometric ratios) to generate...generate useable data. What’s more, measuring and quantifying FRET is often easier said than done. Fortunately...overexpression of fusion proteins it requires can lead to aberrant subcellular localization of the studied PIPs. ...protein construction, cellular expression, and quantification (Kerppola, 2006, 2013). These are described... supraphysiological expression levels. BiFC quantification This is where BiFC outshines FRET — BiFC experiments...BiFC experiments. The ideal control is a mutated variant of one or both of your PIPs in which the protein-interaction...requires that you empirically test a number of mutants to identify those that inhibit BiFC in your cellular...