...for staining. To address this, the membranes are incubated before staining in a buffer containing nonfat...in the presence of a reducing agent, typically containing thiols, to cleave disulfide bonds. Load the samples...variety of tissues to demonstrate that the protein staining matches the expected expression patterns across...level of validation ensures that the antibody is staining the intended target.  Once you have chosen an ...used by the vendor for validation could contain unintended cross-reactive proteins. Similarly, if you vary...with a good overview of the western blot. When planning a western, take care to include the proper positive...

...and cloning sites for inserting the guide sequence (pSpCas9(BB)). The 2A-GFP allows for screening of transfected.....From moving office locations three times, to opening our UK office in 2014, to starting our viral vector...virus, making it ideal for CRISPR pooled library screening experiments. Find lentiCRISPR v2.(If you’ve peeked...  Find the scrambled shRNA vector. pLKO.1 - TRC cloning vector - This vector from the David Root lab is...3rd generation lentiviral transfer plasmid for cloning and expressing new shRNA sequences. For more information...slowly than standard plasmids. Find pLKO.1 - TRC cloning vector. Tet-pLKO-puro - Tet-pLKO-puro contains ...

...mutated methionine was not enough to prevent alternative initiation, so they mutated the methionines at position... You can use DNA sequencing or other molecular cloning techniques to determine CRISPR/sgRNA efficiency...homology directed repair (HDR) and non-homologous end joining (NHEJ). In these reporters, editing typically results...H2B-GFP. This visual read out allows for easy screening of different gRNAs by simply changing the gRNA...transfected cells will express RFP while cells containing the reporter gene that was cleaved by Cas9 and...start site initiation, the lab removed the native methionine start site. This first iteration was named GEmCherry1...

...the next step in high-throughput, genome-wide screening,” Konermann said. “If you want to activate all...her colleagues demonstrated the new system by screening for genes that, when turned on, make melanoma ...CRISPR library enabling genome-scale, knockout screening). As the Zhang lab's Nature paper reports, “Here... can facilitate genome-scale gain-of-function screening when combined with a compact pooled sgRNA library...validation of the top 10 hits.” Two-way CRISPR screening In October, Jonathan Weissman’s lab introduced...developing sgRNAs that will enable even more fine-tuning of gene expression levels and on software for analyzing...Villalta JE, Chen Y, Whitehead EH, Guimaraes C, Panning B, Ploegh HL, Bassik MC, Qi LS, Kampmann M, Weissman...

...consisted of two plasmids: the packaging plasmid containing all the structural genes necessary for the production...retrovirus into the packaging cell line PA12 (containing the psi deletion). A double crossover event between... LTRs, the ψ sequence, a part of the env gene containing the rev response element (RRE), an internal promoter...transfer vector plasmid), 2) the HIV-1 genome containing all viral genes with the exception of the env...distinct expression vectors to target cells - one containing the gene of interest (pTet-IRES-EGFP, pPRIME-...bottleneck for your experiment. Single vector systems containing both your transgene and tTA/rtTA (pINDUCER20,...your institution’s Biosafety Committee before beginning any work. Lentiviral production and transduction...

... the cellular DNA. Fluorescent DNA staining is another DNA staining alternative. This method uses DNA ...proper training and guidance to the people working in the lab facility. This can be done by training eligible...this task, you can use PCR based detection, DNA staining, fluorescent tagging, agar plating, etc. Be wise... is added to the liquid or semi-solid medium (containing nutrients essential for mycoplasma growth). Infected...facility periodically. The CO2 incubators, used for maintaining cell lines, must not be overcrowded (filled no...contamination. Be aware and cautious rather than learning it the hard way! Many thanks to guest blogger...

... a plasmid? Or how much time you have to spend cloning before you can start your experiment? What about...order? Sometimes, it feels like an eternity of cloning, waiting, and repeating before you can finally ...course, these were all 2018 numbers, and the intervening five years, inflation has occurred. Here at Addgene...in science – the time spent by the scientists designing and performing the experiments. This cost varies...and money across the board. Consider alternative cloning strategies There are many strategies beyond the...well) plate! How much do repositories help?   Designing precise calculations for any of the numbers in...Addgene Blog 5 factors to help you choose the right cloning method Find blog posts about scientific sharing...

...found that many researchers feel they lack formal training in a variety of transferable skills. At Addgene...puzzling through your broader research questions and designing future experiments. Being able to work through...at work.   While I was in graduate school and beginning to research non-academic careers, I always felt...dozens of career advice articles, I settled on defining problem solving skills as: One’s ability to logically...the process, you might need to go back to the beginning, reassess the situation, and try a new strategy...scientific method is a core part of our academic training.  Developing strong problem solving skills is ...challenges we encounter. As you spend time in a lab learning techniques and taking on independent projects,...

...can also affect patterning and in some cases even override genetically encoded patterning. Based upon trends... Bioelectric states can alter cell types and patterning There is mounting evidence that signaling via... even be necessary and sufficient for certain patterning activities of entire tissue types or even organs...Since bioelectrical signals are controlled by the opening and closing of ion channel proteins, they are not...developmental biologists and cancer researchers are beginning to turn their attention to bioelectricity. One...Transmembrane voltage potential controls embryonic eye patterning in Xenopus laevis." Development 139.2 (2012):...

...channelrhodopsin-expressing neurons, the channelrhodopsins respond by opening and allowing an influx of ions into the neuron ...s lab and Viviana Gradinaru’s lab used machine learning to design new channelrhodopsins that are more ...channelrhodopsin that’s successful in vivo. Using machine-learning to overcome channelrhodopsin design challenges... Arnold and Gradinaru Labs developed a machine-learning model to help them construct “designer” channelrhodopsins...the library to 1,161 sequences. Thirty of these remaining sequences were selected for characterization with...mice that received ChR2(H134R) did not turn. Left-turning behavior in ChRger2-treated mice ceased when the...Mackey ED, Gradinaru V, Arnold FH (2019) Machine learning-guided channelrhodopsin engineering enables minimally...

... the tides may be turning. In 2013, the NIH announced its first round of Broadening Experience in Scientific...achieved tenure-track positions within 5-7 years of earning their PhDs (3), showing that the ‘standard’ pathway...Scientific Training (BEST) awards, which are designed to support new approaches that address the need to... School. Graduate education programs are also beginning to address these issues independently; for example... may look like. The panel discussion on Friday morning will be dedicated to Academic data and the Labor...PMC3342243. 4. Mathur, Ambika, et al. "Transforming training to reflect the workforce." Science Translational...

...combined with. Combining a PSAM with the IPD of a cation channel, such as the serotonin 3 receptor, will...understand the complex wiring of the brain and the underpinnings of neurological disease, neuroscientists need...a popular tool in the field as it provides a noninvasive method for manipulating cell activity, especially...provided by chemogenetics are instead that it is noninvasive and has longer-lasting effects. Building your...issues can be mitigated by using low doses and running appropriate controls (Jendryka et al., 2019), they...promote depolarization in neurons. Alternatively, combining with the IPD of an anion channel, such as the ...

...vector”. Cloning Method Choose from: Gateway cloning, gene synthesis, Gibson cloning, ligation-independent...ligation-independent cloning, restriction enzyme, TOPO cloning, unknown, or other. Bacterial Resistance Enter...possible, we propagate plasmids in the standard cloning strain DH5α. For plasmids with highly repetitive...plasmids, choose the NEB Stable strain. For plasmids containing the ccdB gene, such as Gateway vectors, choose...

..., I have conducted informational interviews with nine Addgenies. There are a few patterns throughout the... Graduate school: Do I or don’t I? I asked the nine Addgenies about why they did or did not go to grad... had always been a student and I truly enjoyed learning.” Addgene’s Director of Product Management Eric...feeling like I could read a scientific paper from beginning to end and really get it.” On the other hand, ...don’t get your PhD you’re set up for failure. Listening to Kenkel’s story has helped me realize that is... see where my life will take me. I’m also just learning. I have a huge interest in data science,” which...they work at Addgene. I posed this question to all nine of my interviewees. Fabienne Furt, Addgene’s Associate...

...maximum genetic heterozygosity in the population, meaning they more closely mimic the genetic diversity of...be engineered by inserting a targeting vector containing the gene of interest into a zygote, or into embryonic...microinjection of DNA or infection with a viral vector containing the gene construct can be used to insert the ...knockin mice. Co-injection of a targeting vector containing homologous ends to the breakpoint promotes homologous...engineering favors DNA repair via non-homologous end joining, the newly inserted gene may have errors introduced...creating the floxed gene segment. Crossing of mice containing a floxed gene to a Cre-expressing strain (which...post of this series, I’ll discuss crossing and maintaining a mouse strain. Additional resources on the...

...visualization of both organic tissues and cell cultures, opening biological interiors to many inquisitive scientists...select the optimal dye or fluorescent ligand when designing an experiment. You may find yourself defaulting...Fluorescent dyes utilize these fluorophores by combining them with a molecular species with high affinity...affinity for an endogenous molecular target, meaning the dye will be attached to the target through binding...so extra care must be taken to assess if proper staining is achieved. Fluorescent ligands have more flexibility...) can be stably combined with nanoparticles, maintaining intensity at ~85% of its original value after...brightness and high degree of overlap. Dyes for DNA/RNA staining should have high affinity and specificity to nucleic...

... nearly every experiment requiring AAV. Before joining my lab, I had never worked with AAV, so naturally... Two to five days after transfection, the AAV-containing cells and media are harvested and purified. In...duplicated regions like the ITRs. Restriction digest screening for ITR deletion is still necessary. To save time...overlaid. Following ultracentrifugation, the AAV-containing 40% fraction is collected and buffer exchanged...titering by qPCR quantifies the number of genome-containing viral particles that are present in an AAV prep...standard curve, which is generated from an ITR-containing plasmid. This whole process takes ~3 hours to...expression in mammals Use the PEI calculator for planning AAV packaging transfections Find tips for a first...

...were used in this study.  The zCas9i cloning kit is a plant cloning kit that contains a series of vectors...Mapping antibody escape in SARS-CoV-2 by mutational scanning By Seth Kasowitz As multiple antibody-based vaccines...cross-reactivity, antibody candidates underwent affinity screening to select for slow off-rate binders. The best ...protein VP1 is co-expressed with a fusion protein containing VP2C and a protein of interest (POI). The VP2C...with more on the way. S5E2 regulatory element containing vectors developed in the Jordane Dimidschsteind...provide expression selectively in Parvalbumin containing cortical interneurons. We now provide ready-to-use...

...you should always ensure nothing is covering it. Turning on the UV light 3-4 times a week for up to half...microbe-laden aerosols that may enter the hood. Planning your experiments thoroughly before entering the...solves most problems before they occur. With proper planning, you’ll know exactly what materials you’ll need...changes in temperature (especially if you have air conditioning on) so leave them in a sterile incubator. This...make up your master stock, while you can use the remaining vial to grow and freeze additional cells that ... Universität Berlin, Germany. She will soon be joining her PhD lab at the Max Planck Institute of Colloids...the Addgene Blog The Challenges of Cell Culture Cloning Mammalian Cells with the Agarose Method Plasmids...

...Neurological Surgery at the University of Miami. The beginning of this century has seen some major advances in...originally named orthogonal-plane fluorescence optical sectioning (OPFOS) (2). Arne Voie, David Burns and Francis...confocal microscopy In confocal microscopes, optical sectioning of a specimen is based on discriminating the ...to photobleaching.  Furthermore, point-by-point scanning along the entire specimen makes imaging too slow...photo-bleaching or photo-damage than in conventional laser scanning microscopy . Since image acquisition is performed... of imaging rodent brains and spinal cords by combining Ultramicroscopy with tetrahydrofuran-based tissue...Leischner, A. Schierloh, N. Jährling, C.P. Mauch,  K. Deininger, et al. (2007). Nature Methods, 4, 331–336. Pubmed...