... beneath it. You can jump to the different time stamps in the description to watch any sections you’re...watching the whole livestream for a more holistic understand of reproducibility issues and their potential...limits of knowledge as they develop new ways of understanding how the body works, generating new therapeutics...have intricacies that aren’t always mentioned in static written protocols. It is therefore a bit ridiculous...reproducibility is making sure that others can understand your analyses and repeat them. Companies like...initial hypothesis through validation are easier to understand. The need for great stories has, however, made...experiments with too few replicates leading to suspect statistical significance (you can read more about this problem...

... our scientific support staff receives – in fact, the blog originally started as a way to easily store...Thus, we are constantly updating our content to reflect the newest breakthroughs and standards. It’s always... be surprised to learn that Addgene’s mission statement extends beyond plasmids, AAVs, and antibodies ...in the spring of 2022. The first step was to understand information our guide needed to have. Through...someone new to antibodies might need to successfully start using them at the bench, along with questions we...Making content is an iterative process. We are constantly reviewing the resources we currently offer to...

...repeating your work or making similar mistakes in the future. Stay organized Set up a table of contents...computer at the command line, and so was really starting from the beginning. Luckily, a fellow graduate...that it is helpful to those of you just getting started. The practices suggested here may seem tedious ... the analyses: Goal: Before you begin, briefly state the goal of the analysis, including background on... you to perform this analysis. Having a clearly stated, specific goal for each analysis will help you ...computer and is just another name for a folder. For instance, your Desktop directory contains the folders and...code does are very useful for those that are just starting out, and helps clarify the role of the code. ...

... microorganisms, yeast have both a stable haploid and diploid state which is useful for genetic analysis...will catch you up on the core replication and resistance features of yeast vectors and explain how they...the yeast chromosome; however, they tend to be unstable and may be lost during budding. Yeast Centromere...single copy. Unlike the ARS vectors, CEN vectors are stable without integration. Yeast Episomal plasmids (YEp...natural yeast plasmid) allows for 50+ copies to stably propogate per cell. The copy number of these vectors..., due to high rates of spontaneously occuring resistant mutants and the insensitivity of yeast strains...auxotrophic markers. The MX series of antibiotic resistance cassettes is most commonly employed, with the...

...sample into the appropriate stab culture Grow the stab culture Ship the stab culture to the proper location... track the location, quality, growth, and legal status of thousands of plasmids a day, like we do at Addgene...some require going back two steps, some require starting over entirely, and our software needs to handle...corresponding entry in your database. Enjoy your happy lab staff. Barcoded materials make everyone's life easier... tricky procedure failed and one of your techs started over with a new tube. In the first example we’... valid technical reasons to choose a non-Python stack, but if you're working with scientists it's unlikely...

...other references to scientific literature to substantiate the facts upon which it builds. This means you...to relevant primary research articles. Then just start reading and following citations through the literature...Reading primary research literature The best way to understand scientific papers is to practice reading them...give you just enough information to allow you to understand the research question that will be addressed....Is the data of sufficient quality (error bars, statistics, clarity in images etc)? Did they perform all...publication, it doesn’t mean that there are no mistakes or misinterpretations. After all, the science ...research area. Set a goal for paper-reading, even starting out at 1 paper/week will be useful. Check out ...

...immunogenic proteins generated Stable integration into host genome and stable expression of the transgene... generations of lentiviral vectors have been established so far, with safety increasing in each generation...stomatitis virus G protein (VSV-G) that improves the stability and broadens the cellular tropism of the viral... In this case, your gene is repressed in steady state and is expressed when Dox is added to the culture...particles expressing proto-oncogenes or oncogenes for instance should be manipulated with care. See the NIH’s...well. Quickly after transfection (4-8h), cells start releasing viral particles into the culture supernatant...found very effective for T cells transduction for instance. I hope you will find this information useful...

...you need to start a whole new blot? Never fear: membrane stripping is here! Rather than start over, you...whole new gel and membrane transfer. Let’s get started! Before you begin In the best case, you know ahead...stripping and reprobing, maximize your signal by starting with the least-abundant protein and working toward...antibodies will require harsher stripping to remove, so start with the weaker binding ones first to preserve your... produces colored precipitates that permanently stain the membrane, making that section of the blot unusable... loss. If you are still optimizing your system, start with a mild stripping solution and move onto a stringent...incubation times, or try the stringent solution if you started with the mild one. Reprobe Once your membrane is...

...inbox. Best uses Staying up-to-date on new publications in your field. Feeds are constantly updated, so you...challenge is finding relevant papers. The latest installment of the Early Career Researcher Toolbox will highlight...articles related to your actual interests while also staying on top of the ever expanding body of biomedical...characters! Using Twitter to find papers  You’ll need to start following relevant accounts in order to turn Twitter...Scientists: Following other scientists helps you stay on top of what others are studying and gives you...keywords. It’s sort of like a keyword search that’s constantly updated. This Twitter list contains >300 Twitter... papers are published, similar to Facebook or Instagram. Key features Setting up a personalized feed is...

...The day has arrived; you’ve painstakingly cared for your packaging cell line, prepped your DNA, transfected...to move ahead with your infection and make your stable cell line. While we’ve all experienced the pressure...transductions should not be rushed into. Before you start any transduction, you should always titer your virus...components such as LTRs, gag, WPRE, antibiotic resistance-genes, or the transgene itself. Many researchers...are used as it eliminates the need for antibody staining. Check Out Our In-Depth Fluoresence Titering Protocol...Another popular approach utilizes the antibiotic resistance genes carried on transfer plasmids. Target cells... one copy integrates, selecting by antibiotic-resistance or single cell sorting based on expression of...

...trials has been outstanding, with almost all patients experiencing sustained and substantial relief from ...clinical trial for SCD in 2022 and Editas Medicine starting a Cas12-based clinical trial for SCD in 2022 as...targeting the diseased allele specifically. Early-stage trials showed a high success rate in clearing misfolded...above! There are many, many more in the earlier stages of the pipeline, ones that have experienced hiccups...diseases, blindness, and diabetes. Directions and obstacles for future CRISPR therapies CRISPR technology ...systemic approaches, has its own unique set of obstacles. Thus far, several targeted therapies have taken...

... embryonic stem cell stage using one of several techniques. The longest standing technique is the injection...Mice are a common model organism used to understand mammalian traits and genetically engineered mouse... to create engineered mouse models at embryonic stages, and the pros and cons of different genome editing...experiments. Inbred mice are particularly useful for understanding a specific effect related to an experimental...positive selection marker such as an antibiotic resistance gene is also included to select for successfully...which is then implanted into a female animal for gestation. Nucleases that induce double-stranded DNA breaks...which is then implanted into a female mouse for gestation. All three can introduce multiple base pair deletions...

...significantly higher than standard “wired” optogenetics but may become the standard in the future. Now that...by guest blogger Derek Simon. The surgeries and standard molecular neuroscience validation experiments ...Light is able to penetrate brain tissue a short distance so ideal implant placement should be about 0.4...significantly increases. Before you get your experiment started, you will need a laser of the appropriate wavelength...channel, Channelrhodopsin 2 (ChR2), exists in several states: closed/light receptive, open, closed/light insensitive...light must be delivered in pulses. An example of a standard stimulation parameter is 20Hz (laser output), ...source itself. Pulse generation (TTL pulse) is a standard electronics protocol not specific to lasers or...

... to all our #DeckTheLab contestants!           View this post on Instagram                     A...December 12, 2022          View this post on Instagram                     A post shared by 草方 (@gracie_fang...take photos, and then share them on Twitter or Instagram with #DeckTheLab and tagging us, @addgene to be...get you out of.           View this post on Instagram                     A post shared by Xi Chen...picture or video for either contest on Twitter or Instagram by December 31, 2022.  Follow @Addgene, use the...and reveal the winning entries on Twitter and Instagram! Happy decorating, all, and to all a good light...

...single stained reference controls to distinguish one fluorophore from another within a fully stained sample...called autofluorescence (you can see this in your unstained flow controls). Spectral flow cytometers include...tissue types varies, it is critical to include an unstained sample for each tissue type you are running in...Compensation or “reference” controls are samples stained with a single fluorophore. They’re required for...look for that exact signature within your fully stained samples. If you are using conjugated dyes, it is...particular difficulty in unmixing if your sample is stained with a PE/Cy7 antibody from a batch that is not...unmixing. Additionally, it is critical to include unstained beads or cells within your reference group to ...

...ready to store or use. They are quite stable and will withstand freeze/thaw cycles and dehydration (Howard...you can also generate rAAVs in your own lab with standard molecular biology tools and tissue culture experience...choose the optimal titering assay, you’ll need to understand how different titers are measured. A TCID50 measurement...method due to the accessibility of instruments in standard labs. When determining titers by qPCR, we recommend... You should always have a high-quality plasmid standard and rAAV reference material of a known titer on..., D. B., & Harvey, B. K. (2017). Assaying the Stability and Inactivation of AAV Serotype 1 Vectors. Human...recombinant adeno-associated virus type 2 Reference Standard Material. Human Gene Therapy, 21(10), Article ...

...because it has the advantage of starting out with a larger initial starting library size (up to 1012 members...challenging when trying to develop antibodies against unstable targets like membrane proteins that may unfold...This selection platform was designed so that any standard lab can quickly select for sybodies targeting ...To find an optimally binding sybody, you want to start out with a large and diverse pool of sybodies that...al., 2018). Find the Sybody Generation Toolbox! Starting with libraries and a biotinylated (for purification...et al., 2018. Ribosome display The Seeger lab starts with a “pre-enrichment” selection step using ribosome...Zimmermann et al., 2020). This step of the protocol starts out with anti-Myc antibodies bound to an ELISA ...

...functional tests. The newest genome engineer sharing the stage with much-lauded CRISPR-Cas9 is DNA-guided endonuclease...NgAgo is faring with users in a minute, but, to start, let's review why NgAgo is in the spotlight and ...difficult” GC-rich regions of the genome, which seem resistant to Cas9 cleavage (1). Both the CRISPR-Cas9 and...sequence might not always be available at an optimal distance from the target site. For example, NGG may not...genome editing applications. So, how does NgAgo stack up thus far? A few weeks ago, I was going through...and fussy to optimize. One reason could be the instability of the supposedly alien 5’-phosphorylated ssDNA...optimizing genome-editing methods in brain cells and understanding brain tumor biology. When not snipping DNA, ...

...customization with strain-specific promoters, antibiotic resistance selection markers, gRNAs, and dCas9 proteins....range of bacteria. Once transferred, Mobile-CRISPRi stably integrates into the genome and is propagated during...Mobile-CRISPRi does not disrupt gene function and is stable and active for greater than 50 generations. ...40-fold across all species measured. Beyond understanding the basic biology of bacteria, Mobile-CRISPRi...Mobile-CRISPRi can be used to identify antibiotic resistance genes. Genes that are targets of antibiotics are often... guides targeting non-essential genes remained constant, with fitness effects more pronounced at 12 doublings...dCas9. Following integration, Mobile-CRISPRi is stably expressed with no selection required and titratable...

...heavy chain, light chain, constant region, variable region   The constant region of the heavy chain..., or antigen-binding region, and the Fc, or crystallizable region. Two immunoglobulin (Ig) heavy chains...by changing certain amino acids to improve its stability. Additionally, the choice of vector can impact... your sample, you’ll compare your samples to a standard curve. This curve is generated using a serial ...protein that is also detected in the same way. The standard curve that is created from the OD and concentration...antibodies to detect proteins is referred to as staining. This panel of antibodies is used to both identify...Similar to flow cytometry, you’ll use antibodies to stain for markers of one or more specific cell types, ...