...under-transferring.  Membrane pore size Membranes come in different pore sizes for different proteins:...to take. If you’re not sure if you need a smaller pore on your membrane, run a double-membrane transfer...membrane, that’s a good indication that the p membrane pores are too large.  Transfer method   Wet transfers ...increases (Figure 3A).  This step is especially important for highly expressed proteins, including “housekeeping...don’t want to skimp on your washing steps! It’s important to let them wash for the full amount of time, ...blot, you may want to consider using a different reporter or, if you don’t need to compare the protein amounts...Electroblot/dry transfer system Different membrane pore size Adjust SDS:alcohol ratio in transfer buffer...

...experiments didn’t provide any information on other important factors such as what is the best promoter to use... was far from an isolated case. Our scientific support team answers hundreds of AAV questions every year...successfully replicate published works. Of particular importance to these efforts is the open sharing of negative...Figure 2.) At the same time, when scientists who support open science, but do not share their own data -...experimental settings. It is a curated set of technical reports on AAV vectors from Addgene and the CLOVER Center...Center, each one submitted by researchers like you. Reports are reviewed by Addgene scientists for clarity ...

... are more likely to consider cover letters an important part of the application. Even within an organization...core skill, considers cover letters to be very important. On the other hand, our app/dev team rarely, if...that organization or group. This is particularly important for labs and nonprofits, who tend to prioritize... research], and have excellent [list the most important skills for the position]. I am enthusiastic about...about [something that you love and is important for the job]. Notice that the intros above allow you ...that they considered cover letters to be more important for higher-level positions. Many did not require...

...genes important for mesoderm differentiation Now, let's turn tinman and bagpipe, two genes important for...protein Groucho, a transcriptional co-repressor important in development. Groucho family proteins are found...Dana Farber Cancer Institute! Sonic Hedgehog, an important regulator of embryonic development The largest...video game character but rather to an extremely important gene in animal development. To understand where... Tinman, 2016). Bagpipe on the other hand, is important in the formation of the visceral mesoderm which...

...tag. The MBP signal peptide allows the NB to be exported to the periplasm of bacteria where it can be secreted...the hPSC genome using AAV1 and contain an EGFP reporter. Expression of the dCas9-KRAB activator or dCas9...promoter. The gRNA piggyBac plasmids include a mRFP reporter. With this system, they could quantify and track...dCas9 targeting plasmids! Hazelbaker et al., Sci Reports. 2020. https://doi.org/10.1038/s41598-020-57500...Find the plasmids at Addgene! CRISPR RNP electroporation and AAV donor infection (CRISPR-READI) enables...GRABNE) sensors allow for sensitive and high spatiotemporal measurements of norepinephrine. These norepinephrine...

...FLP-FRT. In addition, a variety of means to spatiotemporally control FLP and Cre expression have been developed...These differences in recombinase efficiency are important to keep in mind when deciding whether to use the... only occur at a specific time point? To allow temporal control, ligand regulated Cre and FLPe recombinase...control of Cre recombinase. After in-utero electroporation, this plasmid mix results in sparse labeling... brain area can be transfected by in utero electroporation. To achieve sparse labeling of individual neurons...instance) can be controlled with FLP-FRT. For further temporal control, Tamoxifen inducible Cre versions can ...

...valuable lessons along the way. We think it’s so important that we have written plenty of articles on building...successful mentor relationships, asking for mentor support, and choosing your mentor. What isn’t discussed..., or a graduate student. This is the person you report or talk to on a daily basis who will help you get...bench mentor-mentee relationship is extremely important – without a good set up it could be very difficult...mentor learns where you are at and the level of support you need from them, and you can understand the ...Be My Mentor? Finding and Asking for Mentoring Support   ...

...Joanne as she answered questions to provide moral support and to monitor user replies, but I can’t thank ... basic questions and to provide a small bit of support (you are not alone!) for those going through the...see graduate and postdoc programs beginning to support diverse career path development--but the science...Finally, as I said in the AMA, it’s incredibly important to find good mentors and incentivize good mentorship...and your resources for science communication opportunities like an AMA. One of the best things an early...before, but I also highly recommend taking every opportunity you get to speak publically about your work (...

...of Different Methods for Generating iPSCs. One important note: in this table, reprogramming efficiencies...high is based on the reprogramming efficiencies reported in González et al. A percentage is not listed ... be used to package reprogramming factors. An important milestone of reprogramming is the silencing of...cells for the presence of transgenes is still an important control. Additionally, Sendai virus doesn’t rely...comprised of OCT4, SOX2, LIN28, NANOG, and a GFP reporter. Minicircles are removed from cells by dilution...Characterization Once generating iPSC clones, it’s important to determine how stem cell-like they behave. iPSCs... iPSCs Find gene-specific plasmids (including reporters and RNAi) for:  NANOG  OCT4 SOX2 MYC KLF4 LIN28...

... optogenetics is renowned for enabling precise temporal and spatial control. Optogenetics uses genetically... on and off gene expression while imparting spatiotemporal control, reversibility, and repeatability. ...system, Ye’s lab could edit a tdTomato fluorescent reporter gene in mice using Minicircle DNA vectors encoding...codon, TAG. (To learn more about site-specific incorporation of amino acids using pyrrolysl tRNA synthetase...seen in the figure below. Next, by using a dual reporter fluorescence assay, Hemphill et al. demonstrated...) A Singular System with Precise Dosing and Spatiotemporal Control of CRISPR‐Cas9. Angew Chem Int Ed 58...Rauen RR, Kahn SI, Tsang M, Deiters A (2020) Spatiotemporal Control of CRISPR/Cas9 Function in Cells and...

...Chapter 4: Crystal's approach to management and the importance of realizing that different people have different...learning from peers, and being receptive to those who report to you. Chapter 5: The types of science that Crystal...projects, and how she sets metrics for success. The importance of thinking about the big picture and not just... who are planning on working in industry. The importance of recognizing and leveraging your unique skills...skills and how they've helped her at Novartis. The importance of mentorship in developing career skills. Outro...

...of interest and co-expressing a cytosolic ZtSH2 reporter, they observe fluorescence localizes to the plasma...And orthogonal sensors with different ITAM/SH2 reporter pairs allow multiplexed measurement of different...plasmids here!   Farahani PE, et al. pYtags enable spatiotemporal measurements of receptor tyrosine kinase signaling...10.1101/2022.08.13.503850.  iGECInano NIR calcium reporter Near-infrared (NIR) fluorescent proteins promise...photochemical characteristics than previous NIR calcium reporters iGECI and NIR-GECO2G, and its small size (~1500...sensors Whatever your analyte of interest, it’s important to choose a biosensor that has a binding affinity...

...the mitochondrial calcium uniporter (Martell et al., 2012). It is important to note that the biotin-phenol... of exogenous biotin allowing for more precise temporal control.  Find plasmids for TurboID and miniturbo... where two proteins are fused to complementary reporter proteins that can only assemble when in close ... ascorbate peroxidase as a genetically encoded reporter for electron microscopy." Nature biotechnology...proteomics approach to monitor the composition of spatiotemporally defined protein complexes." Nature communications...

...infection, including both common infections and opportunistic infections not usually seen in healthy individuals... genome had been excised, leaving only a small portion of the LTRs joined together. Clonal Cas9/gRNA-expressing...populations were also immune to HIV-1 reinfection. Importantly, no off-target effects were detected. Kaminski...patients. Although this is an exciting finding, it’s important to note that these HIV-1 patients were ART-naive...Cas9. While these results are discouraging, it’s important to note that these experiments were done in cell...propagate and infect adjacent cells. It will be important to determine if CRISPR/Cas9 escape occurs in an...

...optimized, it’s important to ascertain how long the rescue phenotype will last, and more importantly, if it does...genetic disease in a postnatal mouse model, an important proof of concept for future preclinical and clinical...does not cause obvious germline modification, an important finding given the controversy associated with ...great step forward for CRISPR gene therapy, it’s important to realize that DMD is a simpler case than other...PMID: 26721684. 4. Reardon S. Gene-editing summit supports some research in human embryos. Nature. 2015 Dec...

...the 1980s, is one of the most important ways to spatially and temporally control gene expression, especially...ten colors (see table to the right); in various reports, Sanes and Lichtman have observed from 90-160 distinct...undergone recombination. This default state was disproportionately expressed by a majority of neurons, limiting... 8 colors. The use of AAV provides spatial and temporal control without the need for germline modification...refinements of the Brainbow technique will provide important insights into the complicated physical organization...

... Impact of Non-Elite Journals” found that the proportion of highly cited articles appearing in lower-prestige... to all articles, Anurag Acharya and colleagues report, the percentage of citations to articles in non-elite...publications. (As an interesting aside, Nature recently reported that the most cited work in history is a 1951 ... the best way to measure impact? As that Nature report also noted, papers detailing Nobel Prize-winning... for the impact of research" (2013) Scientific Reports 3, Article number: 1649 doi:10.1038/srep01649 ...

...all the opportunities you can to be communicating about science with others. If opportunities don't exist... not stack the deck in your favor? Look for opportunities to practice transferable skills in ways that...something big like plan a conference Recruit many supporters and mentors that will speak for you (and you ...get real feedback Write whenever you can, find supportive editors so your first drafts are always excellent...

...stage-specific promoter and design a UAS reporter construct, the reporter will only be expressed where and/or...UAS to examine the expression of reporter genes, or to combine reporter expression from one promoter with...the Q system from a gene cluster in the fungus Neurospora crassa. The QUAS promoter is less leaky than ... transcription from the UAS promoter. This is important because it allows them to be used together for...

...useful for genetic engineering approaches. Most importantly for this technology, however, is that the transposase...should be tested in the cell line of interest. Importantly, by varying these ratios, you can either maximize...obstacle, a positive selection cassette can be incorporated, and used during the differentiation process...homologous recombination, which results in the incorporation of your specific edit into the host genome. ...transposon sequences.  The use of PBx is particularly important to avoid any possible random re-integration from...lines, most likely due to DNA toxicity. More importantly, unlike Cre recombinase, the PBx is highly specific...