...to address the comments to “you” rather than “the authors.” By addressing the comments to “you” I can ... figures). After finishing my section-specific comments, I re-read what I wrote about the paper after ...in the first (and clunky) version of my general comments and adjust them if my opinion changed after this...that there is another human who is reading your comments. Receiving a peer-review is already a humbling...young scientists that are reading your peer-review comments will learn from them and propagate this culture...ratio in Arabidopsis, (2) expression patterns in plants with enhanced sodium accumulation in their roots...

...oligomerization can lead to misleading artefacts in your experiments. Read this post from Erik Snapp, Associate Professor...oligomerization can be and how to avoid it in your experiments. Better Dyeing Through Chemistry & Small Molecule...fluorophores can still be used in a wide variety of experiments. Read more to find chemical fluorophores for ...and/or engineer “temporally ordered biochemical events” such as those that occur during development. Read...study basic virus biology while leaving virulent components behind. Minigenomes are stripped down viral genomes...fuel sources.” In her work, Dr. Polka made R-body mutants that respond to different pH’s and some that are...libraries of CRISPR gRNAs to use for screening experiments. Get the full details on how to make these libraries...

...connection between molecular- and cellular-level events and behavior. It’s a problem that he considers ...change has a particular advantage for observing events taking place inside cells that are on the move,...Methods, the FRET changes of these Twitch sensor variants were optimized by testing various linker configurations...large-scale functional screen in bacteria. Sensor variants were then refined by a secondary screen in rat... than intensity-based readouts.” Griesbeck also points out that the approach he and his colleagues took...“trick” to the development of additional color variants. This resulted in the construction of redCALWY...redCALWY-1, a red-shifted FRET sensor for zinc using variants of mOrange2 and mCherry as donor and acceptor ...

...long-lasting way to identify and reference documents, files, or physical reagents. What are persistent identifiers... there is insufficient information about which reagents were used? Unambiguous identification of a reagent...Object Identifier (DOI). Nearly all the papers or preprints you read or produce have one. The DOI serves as...publications help to enable reproducibility of experiments. Additionally, proper citation of these Addgene...standardized persistent identification of research reagents, several journals or organizations now recommend... the RRID and compact identifier formats. Acknowledgments Many thanks to Daniela Bourges for editing ...

...that’s where MTAs, and similar agreements, shine.  These types of agreements allow for centralized tracking...and developments.  MTAs also mean that everyone is on the same page as to how the shared reagents should...organizations to make policy decisions on research use agreements. The UBMTA, which is the MTA most plasmids at...department is responsible for managing research use agreements like MTAs.  Addgene does a lot to ensure that...tracking when biological reagents are shared — both at what time and with whom. Tracking these connections...

...classes, workshops, book clubs, seminars, and social events. Building a community science lab In the Fall...it. We started out by holding a few journal club events and soon realized that it was easy to find people...entrepreneurs using our space to perform early-stage experiments. For example, Dave Grawoig is using nanopore ...sequencing at BosLab to perform proof-of-concept experiments for discovering novel therapeutics. Dave is a...optimizes plant cell culture and transgenesis he wants to share his knowledge with the community by teaching... will engineer yeast to glow, produce synthetic scents, detect compounds in the environment, and openly...ecology but she became a member at BosLab because she wants to engineer microbes. We’ve ordered a few yeast ...

...in silico. These algorithms are trained on vast amounts of data to calculate the relative immunogenicity... replicate the complexity of antigen processing events that take place within an antigen-presenting cell...conditions, etc. that can vary widely between recipients.   In vitro prediction of drug immunogenicity... to validate the findings from the in silico assessments, as well as analyze the contributing factors ...in-vitro models can be built that contain various components of the immune system. Figure 2: In-vitro...Potential Use of HLA Haplotypes to Predict Vulnerable Patients. Frontiers in Immunology, 13. https://www.frontiersin.org...Algorithm Infrastructure and a Vision for Future Enhancements. Frontiers in drug discovery, 2, 952326. https...

...or the use of alternative substrate reagents can permit measurements of luminescence from live cells. Monitor...various species with differing kinetics, substrate requirements and photon emission wavelengths. Renilla luciferase...often used to investigate the effect of regulatory elements, such as promoters, enhancers and untranslated...or the effect of mutations of these regulatory elements on gene expression. The regulatory element (wild-type...measure of signaling activity. Multiple response elements are cloned into a plasmid upstream of a minimal...in luciferase expression and more complex cell treatments can be designed to further explore the pathway...

...cells. Before getting into the mammalian plasmid components, it may be useful to describe the means of introducing... bacterial cells); however, the techniques and reagents vary. Plasmid transfection into mammalian cells...transfection. Unlike bacterial transformation, scientsts do not "select" for cells that have taken up the...transfection and the elusive "mammalian ORI" For many experiments, it is sufficient for the transfected plasmid...plasmid replicates in the cell, as many of these experiments are concluded within 48 h of transfection. Of...void. These ORIs, however, require additional components expressed in trans within the cell for effective...ORIs, respectively. The presence of these viral components greatly reduces the rate of plasmid dilution ...

...competitive endeavor. Due to the COVID-19 pandemic, students will face a reduced supply of internships as a... to the opportunities of their dream.  To help students focus on the application process, and simplify...internships for high school, college, and graduate students. In order to gain a competitive advantage this...errors, sentence structures, and overall format (fonts, font size, legibility, and more). Tips on drafting...leadership status in their current field of expertise augments their authoritative voice. Send a draft of your...in the Chemical Biology Program at Harvard. Roo wants to simplify access to academic resources among economically...outreach among low-income communities to educate parents about resources that can help their children thrive...

...their required components are very similar. While plasmids containing virus-specific elements such as the...or infrequently cycling cells but is fine for experiments with immortalized cell line cultures. Lentivirus...between the two systems, other, more general, viral components such as a heterologous envelope or a post-transcriptional...whole animal administration, such as in mouse experiments. AAV also has multiple serotypes (12 naturally...strategies may need to be considered if size constraints are an issue. Adenovirus Adenovirus has a linear...Addgene blog Viral Vectors 101: Viral Vector Elements Viral Vectors 101: An Introduction to AAV Viral...

... but should contain a table of contents that lays out which experiments can be found in each folder. This...their results. We are adept at recording wet-lab experiments using a “traditional” lab notebook; however, ...the future. Stay organized Set up a table of contents in the top directory: A directory is a file system...document with a brief description of the directory contents, at a minimum. This may or may not be where you...in tact! Take notes Comment in your scripts: Comments on what a given line of code does are very useful...Software You Can Use to Save Time and Track Your Experiments Try out the CRISPR Software Matchmaker Additional...

...Before you jump straight into the exciting new experiments that you’ve been planning, it is a good idea ...yourself have to perform extensive validation experiments for every antibody that you receive; citing existing...However, it is your responsibility to determine what counts as adequate validation. If you are not convinced...your sleeves and perform some extra validation experiments yourself.  Goal of Antibody Validation  As you...assess existing data or plan your own validation experiments, it’s important to know what you are aiming to...expected in another. You’ll need to validate that points 1 and 2 hold true under your specific experimental... that your new antibodies will work for your experiments. Capture MS Strategies This approach uses immunoprecipitation...

...professor who once decided that to teach high school students PCR, they first had to understand hydrogen bonds...every project, time, attention, and/or length restraints mean you will have to pick and choose what concepts...when needed. The trick is to communicate your key points well enough that the audience can take that knowledge...Different modes of communication take up different amounts of mental bandwidth - if not exactly cognitive ... audience, ask! They are experts in what their students know.   Ask the learners themselves for feedback... about the questions you’ve received, read the comments, seek feedback from others, and listen quietly...simpler presentation than I had planned; other times students were fired up and enthusiastic and needed a faster...

...Greek for “self-eating”) is induced by a lack of nutrients and targets cytosol and organelles to the vacuole...double-membraned structures containing undigested cytoplasmic contents. Autophagosomes that have fused with the vacuole...lysosome are trickier to identify because their contents can be at various stages of degradation. Additionally...Atg8p or LC3 doesn’t provide information about the contents of the autophagosome nor does it provide information...sequence. Under normal growth conditions, both variants are excluded from the vacuole and emit red and...the cytosol-targeted and mitochondria-targeted variants are available from Addgene. References 1. Rosado... L. K., Graham, T. E. (2015). Rapid parallel measurements of macroautophagy and mitophagy in mammalian...

...line./ Each color represents a gRNA targeting a specific chromosome. This image represents the composite ...composite of fluorescent measurements from red, blue, and green channels overlaid on a bright-field image. Image...fluorescence from unbound GFP-dCas9. With these improvements, labeling efficiency was similar to that of ... other orthologs. Ma et al. tested their dCas9 variants using gRNAs specific for telomeric sequences and...distance! To the authors’ knowledge, this work represents the first mapping of intrachromosomal loci, a... live cells. However, they note that certain improvements could make CRISPRainbow even better. Adding ...

...washout experiments (Nabet et al. and Weintraub et al.), chemical and genetic rescue experiments (Nabet... for degrading targets in different cellular compartments, and in vivo utility. dTAG molecules will be... line of interest prior to starting knock-in experiments. Test dTAG molecule toxicity in new cell lines...is recommended, while quantitative proteomics experiments will provide the best confirmation of selective...-substrate degradation with dTAG-13. Example experiments See our recent papers noted below for examples...Nabet et al., Erb et al., Weintraub et al.), assessments of fusion chimera functionality (Nabet et al....

...deliver these components together using electroporation. In these bulk cell experiments, Clift et al. ...proteins, and it can even distinguish between protein variants when used with a variant-specific antibody. TRIM21...need to deliver the target antibody. TRIM-Away reagents and reporters are available from Addgene - we ...down the difficulty of your protein knockdown experiments! The TRIM-Away plasmids can be found here. Clift...varying which sgRNAs are used with FIRE-Cas9, experiments can be designed to target multiple loci at once...factor. They then induced cultures of these transformants with various concentrations of estradiol and...

...specific components that are key to the cloning reaction. Understanding the specific components is essential...choosing the correct cloning method for your own experiments, and here we will focus on a unique gene that...on to find out how ccdB can make your cloning experiments a little easier. One of the most time-consuming...choose the most appropriate method for your own experiments. Please check out Addgene’s Choosing a Molecular...these resources further. Feel free to offer your comments and suggestions, and even to submit a guest blog... al. “F plasmid CcdB killer protein: ccdB gene mutants coding for non-cytotoxic proteins which retain ...

... CRISPR-Cas technology is constantly evolving. Variants of Cas proteins can be used for genome editing...In this study, the labs found that anti-CRISPR prevents Cas9 activity in HEK293 by reducing editing from... AcrIIC2 interactions with Cas9 interferes and prevents the correct assembly of the crRNA-Cas complex ...binds to the HNH endonuclease domain of Cas9 and prevents target DNA cleavage (Harrington et al., 2017)....   Five ways to anti-CRISPR proteins in your experiments 1. Reduce off-target effects Prolonged Cas activity...shut off Cas activity? Using inhibitor timing experiments, the Doudna lab found that at least 50% of on-target...vectors. Having a way to shut off CRISPR-Cas systems prevents the off-target effects and cytotoxicity that result...