... the untagged allele. Created with BioRender.com. The first step in creating the plasmids was to identify...be prohibitively expensive for many labs. We’ve created and tested plasmids that use CRISPR/Cas9 to endogenously...first exon of the gene. In this manner, we have created an initial set of 10 plasmids representing 10 human...linker, and the GFP tag sequence to be inserted. Created with BioRender.com. A key feature of our methodology...the double strand break and introducing the tag. Created with BioRender.com. Plasmids, plasmids everywhere...strategy and design described above can be used to create similar donor plasmids for introducing tags into...

... older plates could increase plasmid loss or mutation. Let it incubate Increasing the incubation time ...prep We'll discuss the following techniques to increase plasmid yield. Keep in mind that you might need...particularly stubborn prep. Check the bacterial strain Increase the volume Improve the culture media Let it incubate...more extensive listing of common E. coli strains. Increase the volume Starting with a larger culture volume...you are working with a low copy plasmid. When increasing the culture volume, be aware that a higher volume...origins of replication (e.g. p15A, ColE1) can be increased by adding chloramphenicol to the culture medium...glycerol or glucose (in moderation) may also serve to increase cell density and plasmid yield. Start with a single...

...Laws Bring Increased Postdoc Wages Our most popular blog post this month shares incredibly important information...enacted labor laws will likely increase postdoc earnings in the U.S. This increase, while overdue in my opinion...considering a postdoc. In “Changing Labor Laws Bring Increased Postdoc Wages,” Future of Research executive director...variety of downstream effects including possibly decreasing the number of postdocs various academic institutions...Science Communication Changing Labor Laws Bring Increased Postdoc Wages I hope you all have a great November...

...experiment. Here, we will provide an overview of how to create and use a standard curve and provide some general... I make the standards for a standard curve? You create a standard curve by serially diluting a known control...mix the samples well by vortexing or inversion. Created with BioRender.com.   When choosing a standard... vortexing between each step of the series. To increase the accuracy of the curve, avoid pipetting small...value. How do I plot and use a standard curve? To create a standard curve, you graph the measured response...wider span of concentrations but suffers from decreased specificity, especially as it approaches the minimum... all of the samples in the assay, as this will increase the accuracy of the data. For assays involving...

...enable the creation of CAR-T cells directly within the body. Overall, DIRECTED can increase on-target ...reprogram delivery vehicles like lentiviral vectors, CreVLPs, and eVLPs for in vivo and in vitro applications.... The Ting lab recently made two deposits that increase the utility of this technique.  By engineering... O’Neill The Cohen lab and colleagues recently created a system for recording the transcriptional history...resembling a “ticker tape” recording. The authors created externally-timed bands of color with HaloTag-iPAK4...thin sections of tissue, the Wu Lab is working to create a three-dimensional atlas of protein expression...-95 [K28/43R] (70 µm mounted sagittal section). Credit: Zhuhao Wu, Weill Cornell Medicine.  Interested...

...Jim Woodgett. Public Library of Science (PLOS) created a stir earlier this year when it announced its ...manuscript submission. The rationale includes increasing the ability of reviewers (and, once published...some cases, typically consortia involving genomic screens, the data stream is so prolific that raw information...require access to each individual video. To PLOS’s credit, the organization subsequently clarified what the...and this alone is a reasonable motivation for increasing both data accessibility and methodological detail...Minimally, raw but sufficiently annotated data used to create charts and tables in the paper are in scope. For... up new opportunities for research as well as increasing transparency. Thank you to our guest blogger...

...transdifferentiated in vivo to pancreatic β-like-cells with the expression of pancreatic and duodenal homoeobox... [13]:  Germ cells [14] Hepatocytes [15] Pancreatic β-cells [16] Intestinal tissue [17] Lung epithelial...rare in mammals, an example is observed in the pancreas when excess β-cell damage results in the transdifferentiation...cell types such as fibroblasts, hepatocytes, and pancreatic exocrine cells have been successfully transdifferentiated...differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res, 2009. 19(4.... 38. Thorel, F., et al., Conversion of adult pancreatic alpha-cells to beta-cells after extreme beta-...PubMed PMID: 3690668. 40. Ferber, S., et al., Pancreatic and duodenal homeobox gene 1 induces expression...

... to each to make the library usable for pooled screens. Applications of the cancer pathways ORFs kit Wood...and his colleagues developed the kit for use in screening for pathways that drive resistance to drugs in...the only way to use the kit. “You could use it to screen for pathways that confer any phenotype of interest...drug resistance,” he said. “It could [be used to screen for] morphological features, migration, or survival...use our libraries to do things - sometimes drug screens and sometimes totally different experiments we ...Tracing Read How CRISPR Can Be Used in Genome-Wide Screening Browse 28 Hot Plasmid Technologies from 2015 ... cancer screening...

...Creating great educational science videos is tough. Is the content accurate? Are we conveying complex...people in tight locations. Conference rooms for on-screen presenters! Labs and benches! Everything was out...videos. It’s really nice to have a scientist on-screen, somebody who really knows the protocol and can...would be to use an iPad to shoot.  Build: Jennifer created a mobile camera area that included iPad and her...where I could focus on camera and audio, and the on-screen talent could just focus on their interview or performance...re-opened very early in the pandemic with some incredibly strict safety protocols to keep Addgenies safe...camera) would remain masked, it freed us up to be creative.  Test: Our first official shoot went off with...

...developing zebrafish pancreas. They first tagged these cells using plasmids. Then using HOTcre, a method that...of quiescent and proliferative pancreatic β-cells identified by HOTcre mediated labeling." Proceedings...these systems check out Gerald Rubin’s lab who created a set of modular vectors that can be used to optimally...unicellular organisms, they can also be used to easily screen multiple therapeutic compounds in one experiment...embryos with DNA or RNA to edit their genomes or to create transgenic animals. Zebrafish are an ideal model...β-cells that originate in different regions of the pancreas and produce different levels of insulin (Hesselson...

...Symptoms of AIDS include rapid weight loss and an increased risk of infection, including both common infections...ART-treated patients lack plasma HIV-1, they are at increased risk for other chronic diseases, including dementia...disorders, nerve damage, and cardiac disease. This increased disease risk is attributed to latent HIV reservoirs... used histone deacetylase (HDAC) inhibitors to increase transcription of the integrated HIV-1 genome. ... enhancer region near the NF-KB binding sites increased activation of HIV promoter-luciferase constructs...microglial line, treatment with CRISPR/Cas9 SAM increased the percentage of cells with activated HIV-1, ...cell. ZFNs and CRISPR/Cas9 have also been used to create mutations in genes essential to HIV-1 replication...

...resulting in decreased BRET2 signal. With increased concentration of drug, the BRET2 signal decreases. ...leads to downstream effects.  Bryan Roth’s lab created a collection of plasmids called TRUPATH to interrogate...the complex will disassociate, resulting in a decreased GFP2 emission. The activation state can be measured...showing the effectiveness of using TRUPATH as a screening technique. Lastly, the activity of both antagonists...plasmids are available as a collection, facilitating screening across the transducerome, to gain a more complete...

...scaffold. To increase expression, Tanenbaum et al. modified the GCN4 sequence to increase its alpha-helical...activation. Multiple nuclear localization signals (NLS) increase protein import into the nucleus. In developing... Weissman labs took this biological lesson and created a system to amplify fluorescent signals. Named ...protein is expressed at low levels, you have to increase your imaging time to get enough signal. This workaround...cells without affecting protein function, thereby creating a single-molecule reporter of intracellular processes...alpha-helical structure and stability, creating v4 SunTag. Since the v4 system does not display protein aggregation...

... Figure 1: Initial steps in the CRISPR-pooled screening using sgRNAiBAR. Image from Zhu et al., 2019.  ... Peking University has developed a new CRISPR screening method that makes use of guide RNAs with internal...false-positive rate discovery rate, conventional CRISPR screens require a low multiplicity of infection (MOI) during... annotated human gene, it enables genome-wide screening with a high MOI, which is beneficial for a low... calcineurin dynamics and can serve as a drug-screening platform to identify new antifungals that target... assay, and are not suited to high throughput screening, calcineurin dynamics measurements, or monitoring... attached fluorescent protein to the DNA. They created plasmids for purification of tTALE-eGFP and tTALE-mCherry...

...finding. Transcriptional program changes from this screen successfully differentiated cells resembling all...from Joung et al.)    Putting MORF to work Screens and selections In its current format, the MORF ...library is most immediately useful for TF library screens. Previously engineered TF libraries were not as... library can be used with other selection and screening mechanisms, although the unique barcodes for each...mutation and/or TF dysregulation. A targeted TF screening approach can not only lead to a better understanding...better simulate a disease in a model system. TF screens can be designed to identify custom TF programs ...Cas-9 knockout and transcriptional activation screening. Nat Protoc. 2017 (4):828-863. doi: 10.1038/nprot...

...approach, the Anna Cereseto Lab created pX-evoCas9, which they showed also increased specificity. Most recently...broader PAM specificity can target more sites. Created with BioRender.com. Specificity Specificity...multiple PAM sequences (NG, GAA, and GT), greatly increasing the number of genomic loci that can be targeted...developed SpCas9-HF1. Both of the enzymes showed decreased off-target activity relative to Cas9. Since then...accurate Cas9 (HypaCas9). This nuclease showed decreased or similar off-target activity, relative to eSpCas9..., et al. "Directed evolution of CRISPR-Cas9 to increase its specificity." Nature communications 9.1 (2018...

... complexity of the device increases, as does the price. With this increased complexity comes the need ...adapted and combined with creativity and feedback from the community to create state-of-the-art devices...via workshops, seminars, or hackathons greatly increases accessibility. Through this approach, the Planktoscope...Madagascar. Platforms that allow for on-going creativity with the construction plans. Appropriate open-source...sources within the next few years, with the goal of creating more transparent and reproducible science in the...

...new Antibody Guide and not just because I helped create it. But while antibodies, as the OG (as the kids...reagents we just didn't have the space to give proper credit to. Thankfully, as Addgene's resident blog editor...the majority of these alternative reagents are created in the lab, instead of generated in vivo, they ...Listed in no particular order.) Fab fragments are created through enzymatic cleavage of the variable region...in clinical applications. Fab fragments can be created in lab in situations where a suitable antibody ... fusion proteins similar to a Fab fragment but created through engineering instead of cleavage. They are...cheaper to make than antibodies. Minibodies are created by binding together two scFvs with a CH3 linker...

...antibodies produced from its serum several weeks later. Created with biorender.com. To generate polyclonal ...additional booster immunizations every 2-3 weeks to increase the antibody titer. The titer is tested following...epitopes of the target protein. While this can increase background staining (see next section), it may...experimental protocol, using a polyclonal antibody increases the likelihood that one of the clones will still...crosslinking (ChIP) or fixation (IHC). Because of their increased sensitivity, polyclonal antibodies are particularly...epitopes of their target protein thereby greatly increasing signal amplification.  When to stay away from...population recognizes many different epitopes, increasing the likelihood of cross-reactivity with other...

... Gen AdVs were designed to have increased cloning capacity, decreased  potential for the generation of...E4 are deleted in 2nd generation AdVs, thereby increasing cloning capacity to ~12 Kb. The ever popular ...generated high interest for gene therapy due their increased cloning capacity (up to 36 kb!), long-term transgene...AdEasyTM system is the most popular method for creating adenoviral vector constructs. It consists of 2...can be repeated multiple times (2-4 rounds) at increasing scale over the course of 1-2 weeks. Each round...of amplification should result in a 10-100-fold increase in virus. Purification (2 days) - Purification...