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...use electro-competent cells instead of the more common chemically-competent cells. You should perform, ...This way you can then cut the plasmid backbone as well as the insert with EcoRI and HindIII and, when you...and verification required for the process to work well, so let’s go over the details step-by-step. 1. ...varies for different enzymes. Many companies now sell fast digest enzymes that can digest large amounts...backbone from the enzymes used to digest them as well as any pieces cut out or off of them. An easy way...undigested controls) are loaded at the top of the gel in wells positioned toward the cathode (- charge). When the... manufacturer’s instructions for your competent cells. For most standard cloning, you can transform 1-...

...are commonly found in cells, and without something to block RNase activity upon cell lysis, RNA is degraded... within cells or tissues- they don’t actually chemically stabilize the RNA molecules (Allewell and Sarma...RNases such as RNaseZAP. When harvesting tissues, cells, plants, fungi, or bacteria, keep samples cold and...before RNases had a chance to break down RNA upon cell lysis.  In the late 1970’s, Chirgwin and colleagues...keeping your samples cold until homogenization and cell lysis. This can be challenging depending on your...developed ways to “stabilize” RNA within tissues before cell lysis. These products, namely RNAlater®  from Thermo...is not an issue.  References                 Allewell NM, Sama A (1974) The effect of ammonium sulfate...

...designed an RNA bait containing three well conserved but not well characterized RNA motifs. This bait identified...proteins that bind an RNA motif of interest in living cells. Challenges with existing RNA-protein interaction...and naturally occurring complexes present in the cell, it can also introduce non-physiological RNA-protein...a short timescale, such as during drug action or cell signaling. Limitations of RaPID Like other BirA*... controls include both scrambled RNA motifs and cells transfected with BirA* protein alone (i.e. no RNA...protein that’s highly expressed in neural progenitor cells. Knock-down of QKI decreased Zika viral RNA levels...2018). RNA–protein interaction detection in living cells. Nature Methods, 15, 207-212. PubMed PMID: 29400715...

...protein of interest in cells and tissues. Although antibody production is well established, it’s also .... RANbodies are expressed in 293T cells and are secreted from cells due to their IgGκ signal peptide sequence... sequence, making them easy to harvest from cell culture media. Their His tags allow for further purification...RANBody While all three reporters stain cultured cells and tissue sections, some reporters are better suited... signal amplification of HRP Staining cultured cells and tissue sections Amplifying weak signals of fluorescent...Labeled anti-HA or -Myc antibody Staining cultured cells and tissue sections When detected with a labeled...Labeled Anti-Chicken IgY antibody Staining cultured cells and tissue sections Great for multicolor staining...

...Many thanks to our guest blogger Gary McDowell! Gary S. McDowell is currently the Executive Director of...This post was contributed by guest blogger, Gary McDowell, executive director of Future of Research. On ...opportunity of the lapsed mandate (or are being compelled at the state level, in the case of some public...expecting a salary increase have had that increase cancelled. Rounding out the remainder, 2% of postdocs who... are continuing with their plans, and 17 have cancelled their plans. 2 of those raising - the University...Illinois and the University of Michigan - initially cancelled their plans, but have since reversed their position... In addition, PIs at some of the institutions cancelling plans are going ahead with raises anyway, including...

...indicator. Archon1 can embed itself into the cell membrane where cell voltage can then be measured. Archon1 ... it. Using a robotic picking approach to select cells with variant characteristics they wanted, the team...2in1 vector cloning system Article contributed by Michelle Cronin  Listen to the 2in1 cloning podcast segment...through recombination. The 2in1 cloning system is well suited for protein-protein interaction (PPI) studies...offers a way to spatially and temporally control cellular processes in vivo. Many photoactive proteins exist...approach to control protein-protein interactions in cells because the protein binders can be customized for...photo-reversible protein-protein interactions can control subcellular localization processes in vivo. They deposited...

...Control of Gene Expression. Cell 152:1173–1183 . https://doi.org/10.1016/j.cell.2013.02.022 Sajwan S, Mannervik...and Fluorescence Imaging. Cell 159:635–646 . https://doi.org/10.1016/j.cell.2014.09.039 Zhou H, Liu J,...from when looking to activate genes across diverse cell lines.   dCas9-VP64 Description CRISPR activation...large and are not expressed consistently throughout cells. VPR Description VPR fuses a tripartite complex ...activator used in other CRISPR activation methods, as well as two other potent transcriptional activators (...activation levels than SAM in some loci across Drosophila cell lines (Sajwan et al., 2019). SPH SPH combines components... Gabrielle Clouse...

...protocol, we can prep two 96 well plates in just under 3 hours or one 96 well plate in about an hour. Automating...our set up. The cooling block is used to keep our cells from warming up too much and dying.  Also, we added...added a couple of platforms that fit 48 well q- trays allowing us to do up to 96 transformations in one...simple. By clicking a few buttons you can quickly tell the Biomek FxP how much sample to pick up, where...pick up 96 tips for easy sample transfer between 96 well plates. The second pod can pick up 8 tips and position...be useful if you’re transferring samples from 96 wells plates onto agar q-trays where the samples must ...Have you ever wondered who fills all those vials? Well, it used to be a lab assistant, but with increased...

...complex cellular behaviors requires making complex manipulations to cells. For example: in a cell there ... yield a cellular phenotype, translational output, etc., which can support a larger cellular engineering...TU, or even an individual part, will behave in a cell. For unpredictable cases, MoClo provides two answers...allow you to study natural genetic circuits in the cell. Natural and synthetic product production A major...biofuels, drug precursors, etc., microbially or in cell-free systems. These pathways typically involve multiple... be used to assemble multiplexed gRNA systems as well as additional components such as selectable markers...

...volunteers as well. 4) Blog - Hone your writing skills Take the DIY approach and start a blog. Stellar talents...contributed by guest blogger Sarah Schmidt, a Marie Curie Fellow at The Sainsbury Laboratory. Are you a science ... probably write for your institute’s blog. The Wellcome Trust blog or the John Innes Student Voice are...decide to start your own blog, check out these two excellent articles: On the Origin of Science Writers How...). A great opportunity is the AAAS Mass Media Fellowship, which places science students for 10 busy weeks...American or National Geographic. Similarly, media fellowships from the British Science Association allow you...started opening up. My current job as a Marie Curie Fellow is a direct consequence of a blog that I started...

...for pathogen detection In 2014, as postdoctoral fellows with Jim Collins and Peng Yin at the Wyss Institute...circuits to be deployed out of the laboratory in a cell-free and biosafe format that was stable for up to...possible and submitted our manuscript to the journal Cell just six weeks later. The Zika publication timeline...switch designs, CRISPR/Cas9-mediated genotyping, as well as our electronic reader that provides portable ... demonstrated our technology on active virus, as well as shown that we can detect the virus in a clinically...of Toronto. His research is focused on developing cell-free synthetic biology applications for human health...Virus Using Programmable Biomolecular Components." Cell (2016). PubMed PMID: 27160350. Find Plasmids from...

...alone. At least I could tell that they were mushrooms. In some cases I couldn’t tell if the bags contained...origin of cellular life. His lab has long hypothesized that life started with RNA and a simple cell membrane...Chinese medicine. There were hundreds of stalls selling a myriad of items, only a small number of which...by Nobel Laureate (and Addgene depositor) Craig Mello. Taking his groundbreaking work in RNAi one step...involved in preventing silencing of self-genes. Dr. Mello presented a hypothesis that these pathways are involved... circular RNA they have identified resulted in a cell migration defect. They have created a circRNA website...discover circRNAs in RNA-seq data. As far as I can tell, we don’t really know what circRNAs do yet or how...

...genes should be ‘read’ by the cellular machinery. This is how most cells in the body contain the same ...complex of DNA and histone proteins that compacts cellular DNA into chromosomes and reduces the risk of dangerous...histones changes, then gene expression can change as well. The more frequently and tightly bound DNA is to...the more compacted it becomes. This prevents the cellular machinery from physically interacting with the...function. Angelman syndrome is characterized by intellectual and developmental disability, jerky movements...visualise changes in histone methylation in living cells. Essentially this reporter construct consists of...recognise lysine 27 methylation. When tested in living cells where methylation of these sites was controlled,...

...vitro as well as in vivo in yeast, plant, insect, and mammalian cells, including human cells. piggyBac...is active in vertebrates and integrates in human cells at rates similar to retroviral vectors. The hyperactive...piggyBac has an integration efficiency in human cells similar to retroviral vectors. The hyperactive PB...transposase (hyPB) is ~10-fold more active in mammalian cells compared to the codon-optimized wild-type piggyBac...G/C)TNA. Tol2 can deliver 10-11 kB to mammalian cells without decreased efficiency, with a maximum cargo...and has lower efficiency of integration in human cells than piggyBac and Sleeping Beauty. Minimal Tol2 ... or barcodes. When transposons are delivered to cells or a model organism, they insert into the host’s...

...check its subcellular location and solubility. If it’s membrane-bound or located in a subcellular compartment...reagent. Be aware that this approach requires excellent attention to detail, since you’ll end up with ...question before running one.  Good on gradients? Excellent! Now, let’s head west, intrepid reader! Protein...strengths. If that’s not working, consider using a subcellular fractionation kit or a lysing protocol developed...samples and reference ladders are not next to empty wells. Most people will simply load an extra ladder or...replicate (ie, leftover sample) to fill the relevant wells. Similarly, if you load a gel and then forget to...the time with smaller tasks, or simply enjoy some well-deserved down time while waiting for your timer ...

...For about as long as the disciplines have existed, cell and molecular biologists have been continuously ... peer into the mysterious, microscopic world of cells. The development of antibodies as tools allowed ...was the existence of high quality antibodies for well defined peptides. Munro and Pelham’s first published...monoclonals would help the field explore Myc’s role in cell proliferation and tumorigenesis.  Using two different...the same antibody developed almost 40 years ago? Well…kinda. In some cases, antibody providers are distributing... human IgG2, mouse IgE, goat IgG, (and more!) as well as the original mouse IgG1. And the refinement of...specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616. https://doi.org/10.1128/mcb.5.12.3610...

...both SpCas9 and your gRNA in a variety of mammalian cell types. Purpose  SpCas9 This plasmid expresses Streptococcus...the Zhang lab has tested in a variety of mammalian cell lines. Cas9, of course, is the key component of ... puromycin resistance gene enables selection of cells with the plasmid post transfection. The 2A "self...can be used to transfect a variety of mammalian cells and can be modified for viral trasduction (see the...resistance gene that made it less effective in certain cell lines. This point mutation has been corrected in... your gRNA of interest in a variety of mammalian cell lines. Alternative forms of this vector contain ...directed repair. You can find all of these variants, as well as links to the Zhang Lab's Nature Protocols Publication...

...scientists. What works well in one cell or neuron type may not work well for a neighboring cell type, even within...options while AAV8 (82%) and AAV9 (84%) were both excellent at infecting our target neurons (Figure 1). However... conditions (3,5,8), injection titers (1,2), and cell types used for packaging viral particles (6), can...methods and image descriptions, a minimum n = 2, and well labeled high- and low-magnification images demonstrating... vector performance in various brain regions and cell types. Power in numbers A Pubmed search for “AAV....169 Gruntman, A. M., Su, L., Su, Q., Gao, G., Mueller, C., & Flotte, T. R. (2015). Stability and compatibility...

...Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi.org/10.1016/j.cell.2015.09.038 Find plasmids...are capable of cleaving genomic DNA in mammalian cells and zebrafish embryos, and they can be used to modify...domains and allows for expanded PAM recognition as well as increased specificity and lower off-target activity... adapted for targeted RNA cleavage in mammalian cells. The REPAIR (RNA Editing for Programmable A to I...Han S, Joung J, Belanto JJ, Verdine V, Cox DBT, Kellner MJ, Regev A, Lander ES, Voytas DF, Ting AY, Zhang... DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang F (2017) RNA editing with CRISPR-Cas13...reveals specificities of Cpf1 endonucleases in human cells. Nat Biotechnol 34:863–868 . https://doi.org/10.1038...

...blog The blog has many other excellent posts on science career topics as well. Instead of rewriting or coming...everyone, particularly if you’re more interested in selling a product than communicating your ideas with people...has great questions! 2. The r/Science subreddit is well moderated. One of our original worries about sharing...rousing the trolls. 3. The AMA process was simple and well organized. You can check out the r/Science AMA Submission...with our general mission to share- as opposed to sell- science. 4. You can prepare answers beforehand....answers off the cuff, but like to be able to provide well thought out responses. I hung out with Joanne as...small quibble. If you are not too worried about selling yourself and really do just want to get information...