...Whereas Southern blotting is labor intensive and requires a large amount of high-quality DNA, real-time ...automation, higher throughput screening, and a lower requirement for the amount of DNA used saving the researcher...Figure 1). Though some labs may prefer to send DNA directly for sequencing (more on that below), restriction...dideoxynucleotides (ddNTPs), lacking the 3’ hydroxyl group required for the extension of a DNA strand (8). Adding ...whole genomes or profiling transcriptomes (RNAseq) require sequencing of many samples at once for later comparison...confirming the results of a cloning experiment, require only one or a few sequencing reactions and may ...sequencing technologies could one day sequence an entire genome for under $1,000 (10). The future of sequencing...

...Essentially the goal was to determine what data is required for the academic research community to develop...prepare scientists for positions outside of academia Require that postdoc salaries be paid on fellowships instead... joining long programs if it is obvious that a desired career outcome is not easily achieved. Of note...incentives that encourage PIs and universities to hire a glut of trainees at low wages will still be there...take steps to broaden their skills beyond those required at the lab bench. At Addgene we try to do our ...was the idea that simply satisfying a percent requirement for diversity in graduate students, postdocs,...individuals are discluded/not treated equitably even when hired; well-meaning mentors often fail to judge the work...

...CRISPR/Cas9 for targeted genomic deletions We were inspired to produce intrachromosomal deletions based on... advent of CRISPR/Cas9, we began to explore the paired double-strand break (DSB) approach at a variety...applications in which the production of deletions by paired DSBs may be particularly useful. The first is the...context. An alternate outcome to deletions following paired DSBs is inversions [3]. These rearranged segments...loss-of-function frameshift mutations. However these may require restriction fragment length polymorphism analysis...target two exonic sites within a single gene using paired DSBs. This method allows for deletion screening...

...protein products is desired, as multiple selection markers can ensure that all desired tags have been integrated...linear PCR product is introduced into the cell by direct transformation. A given insert will typically contain...Simply design your amplification primers with the desired targeting homology—in frame, of course—and start...ONE 8(7): e67902. Many imaging studies rely on direct fusion of fluorescent proteins (FPs) to a yeast...specific filter sets and experimental output requirements. Select from these yeast-optimized fluorophore...

...Promoter trap systems lack a promoter and thus require the trapping of an endogenous promoter. The system...traps, enhancer traps lack enhancer elements and require the trapping of endogenous enhancers to stimulate...constructs that contain splice acceptors. They require insertion into an intron of a gene, where they ...like promoter traps. While this system does not require insertion into an exon, like promoter traps, it...think enhancer traps are the top trap – they don’t require insertion in a highly specific gene region and ... the gene that they are monitoring as they are directly integrated into it. Enhancer traps do not need...

... may be required for more complex experiments. A multiplexed experiment, for example, requires staining... negative controls Most biological experiments require positive and negative controls to ensure proper...is generally a sample or group that will have a desired response. For example, if you are studying the ...sample, the antibody binds specifically to its desired target (purple) and nonspecifically through the...The Preimmune serum control will not bind to the desired target but will bind nonspecifically through the... site.      Secondary antibody specificity  In indirect staining approaches, secondary antibody specificity...

...), and photoswitches. Light-gated ion channels directly open and close when exposed to light and change... photoswitches OptoXRs are great for activating entire signaling pathways, but what if you’re interested...potential, then a channel with fast kinetics may be required, whereas a channel with slower kinetics can be...Principles for applying optogenetic tools derived from direct comparative analysis of microbial opsins. Nature...., & Bamberg, E. (2003). Channelrhodopsin-2, a directly light-gated cation-selective membrane channel....BiPOLES is an optogenetic tool developed for bidirectional dual-color control of neurons. Nature Communications...

...Direct reprogramming describes the process where differentiated cells are turned into a cell type of ...Though a valuable tool for regenerative medicine, direct reprogramming is an inefficient process, with the... the majority of cells failing to develop the desired identity. The development of single cell technologies...CellTag The Morris Lab developed CellTag to track the direct reprogramming of mouse embryonic fibroblasts (MEFs..."Single-cell mapping of lineage and identity in direct reprogramming." Nature 564.7735 (2018): 219. PubMed...pluripotent stems cells (iPSCS) and reprogramming and direct reprogramming Explore different methods for delivery...

...come up with a great experimental plan that requires indirect staining of both targets in your specimen.... are the same isotype and therefore cannot be indirectly probed simultaneously on the sample. What to ...matter? Isotype matters when constraining using the indirect method since a secondary antibody reactive to ...In the case above, you can’t costain with your desired antibodies because the pair belong to the same ...the same isotype can not be used for multiplexed indirect staining methods since the secondary antibody ...

...into primary cells, so most primary cell lines require viral infection. A new paper from Niels Geijsen...they could simply induce cells to take up protein directly? It’s not a new idea, but it comes with a few ...expression of a luciferase reporter. CPP was not required for protein entry into cells! After verifying ...non-detergent sulfobetaine 201 (NDSB-201) were required for protein delivery. D’Astolfo et al. coined ...take up, and continued protein expression would require multiple transduction events. For these reasons...concerns associated with virus work. Fourth, working directly with proteins removes the limitations of packaging...

...are required for the transduced cell to become light responsive than fluorescent proteins required to ...always be enough and therefore higher titer will be required. For example, retrograde AAV gets transported ...with your experimental setup. Some experiments require higher gene expression and therefore higher titer...if you end up concentrating your AAV above your desired titer and want to dilute it, err on the side of...less buffer than you would to achieve the exact desired concentration to avoid over-diluting, and make ...

...enabling genome-wide interrogation of the genes required for cellular processes. For a successful screen...plaques. ApoB bound lipoproteins (ApoB-LPs) are indirectly measured through their triglyceride or cholesterol...strong correlation with cardiovascular disease and indirect measurement of ApoB-LPs through triglyceride/cholesterol...et al, 2017).  In an effort to measure ApoB-LPs directly and develop a potentially high throughput in vivo...transcription. Transcription from σ54 promoters requires binding of bacterial enhancer binding proteins...difficulties such as small fluorescence changes or a requirement for very high excitation light intensities have...

...of the Nikon Imaging Center at UCSF. A common requirement for live cell imaging experiments is the ability...simultaneously. To do so with fluorescent protein labels requires multiple fluorescent proteins whose excitation... means that the choice of fluorescent proteins requires some thought. The first step in choosing fluorescent...fluorescent proteins can be separated from each other requires knowledge of their excitation and emission spectra...Beware that these infrared fluorescent proteins require biliverdin as a cofactor and so you may need to...Thorn is an associate professor at UCSF, where he directs the Nikon Imaging Center. He received his PhD in...

... interested in using GCE, here are the basic requirements of use. First, and most obviously, you need ... GCE are essentially those needed to make site-directed mutagenesis, many researchers new to GCE aren’...field, it’s not uncommon to find protocols that require reagents which aren’t broadly or easily available...complicated.” And Ryan Mehl would know. He’s the director of GCE4All, an NIH-funded center located at Oregon...phosphoserine and, because it uses ∆serB strains, does not require exogenously added phosphoserine. A community resource...using, GCE to reach out to them and let them know directly what protocols would be useful. Think GCE could...

...accessible by Cas9. Our previous blog post The PAM Requirement and Expanding CRISPR Beyond SpCas9 described ...a rationally engineered Cas9, SpCas9-NG, which required only the di-nucleotide PAM NG (Nishimasu et al...for base editing techniques, where Cas9 must be directed to a very specific site. Specificity vs. sensitivity...of different sites. Using a combined random and directed mutagenesis approach, the Anna Cereseto Lab created...specificity. Most recently, the Jungjoon Lee Lab used directed evolution to generate Sniper-Cas9 resulting in...Central PMCID: PMC5951633. Lee, Jungjoon K., et al. "Directed evolution of CRISPR-Cas9 to increase its specificity...

...maps have the identities of most features labeled directly on them making them much easier to quickly identify... see above) for your own reference or to paste directly into your notebook. This will make it easier for... immediate, high-level analysis, you can click directly on the “Analyze Sequence” button to pull up an...sizes, colors used to indicate them on the map, direction (if relevant), and types. Types listed are standard...sequences, binding site locations, lengths, and direction. Clicking on a particular primer here also highlights...Addgene software development teams for working tirelessly to implement these improvements. Let us know ...

...I like to envision that my research will have a direct impact on healthcare and the community. As a result...like-minded people, I quickly realize that a company requires more than just good data to become a product. ...settings, etc. What project in the Collins lab inspired Sample6? The initial work focused on using bacteriophages...graduate school usually) is one of the key skills required to achieve success in this business. A good presentation...expenditure of effort in preparation and practice is directly related to success presenting." A journey ...and succeeding. Take home Starting a company requires dedication, passion, and around-the-clock work...

...recognition sequence, they can be customized to direct assembly of DNA fragments. When designed correctly...contains sites with complementary overhangs that direct assembly of the final ligation product. As shown... be re-digested. In contrast, formation of the desired ligation product is irreversible because this construct...a single reaction! These overhangs specify the desired order of fragments, and the loss of enzyme recognition...Exonuclease-based methods like Gibson assembly require 20-40 bp of homology at the ends of DNA fragments...cloning is not 100% sequence-independent: to avoid undesired digestion, the Type IIS site used must not be ...

...distinguish proteins that directly interact with the bait from those that indirectly interact or from proteins...throughout a pulldown step, such as AP-MS. This requirement often prevents the identification of weak and...in a native cell contact. Other PPI methods may require protein interactions to remain stable (or to be...of Split-BioID is its speed of labeling. BioID requires 6-24 hours for tetracycline induced expression...available from Addgene! References Schopp, I.M., Ramirez, C.C., Debeljak, J., Kreibich, E., Skribbe, M.,...

...other. In a direct approach, that is simple - but what about when you need to use an indirect (primary and... and to work in a variety of applications. The desired specificity of your secondary antibody depends ...expressed at very low levels or your application requires a high degree of affinity, then species choice...antibodies may be the better choice for assays that require high affinity binding, such as immunoprecipitations...multiple targets When staining multiple targets indirectly, you must strategically plan primary/secondary...