...Tom Ellis highlights the importance of keeping up-to-date with the literature of a couple of well-known...yet attainable milestones that contribute to intellectual thought and discovery to the scientific community...ideas. What mentors are available for guidance, intelligent discussion, and/or brainstorming sessions to ...increase chances that your project will be fresh as well.” Don't underestimate the importance of choosing...for you.   Acknowledgments Thank you to Dr. Tom Ellis (Imperial College London), Dr. Connie Cepko (Harvard...

...practice (GLP) (important buzzwords in industry) as well as skills in industry positions. There were, however...projects, this can cause conflicts and favoritism, as well as affect morale and cause gossip). These kinds ...that, when those contracts fall through or are cancelled, then redundancies can be made within the company...  Working abroad: Research in Australia I then travelled abroad to work at the University of Queensland...customer service (recruitment agencies often don’t tell you what company a job is with until just before...towards the scientific aspects of the company as well and use my other skills and interests. I was so ...flexibility of a ‘start-up’. I was surprised how well I fit in with the company and that my ideas and ...

...desired mutation in mouse ES cells, select for the mutation and inject the ES cells into a mouse embryo to ...Cas-mediated genome engineering." Cell 154 (2013):1370-9. DOI: 10.1016/j.cell.2013.08.022 PubMed: PMID: 23992847...then electroporate it into mouse embryonic stem cells. After selecting for clones with the recombined ... your chimeric mice did not contain edited germ cells, your experiment would be unsuccessful. CRISPR made... targeting the same or complementary strands, as well as guides facing towards or away from each other...

...professional and personal development, is essential to excelling in the modern world. Talent knows no boundaries...me in my next steps. For example, how to get a fellowship to travel abroad, how to write cover letters,...mentees identified career opportunities in science as well as the developmental needs and resources needed ...the program included improvements in writing fellowships, giving oral presentations, and preparing for...profesional networks, and nominations for government fellowships. The closing and formal evaluation of the pilot...Spanish government’s support for the IMP program as well as for the Spanish scientific community overseas...particularly interested in breast cancer and metastasis as well as teaching and mentoring activities. Follow her...

...protein purification as well. This tag is not suitable for use in apoptotic cells as Caspase3/7 both cleave...originally derived from jellyfish (Aequorea victoria) and can serve as an epitope tag as well as a fluorescent...Addgene’s catalog including a mouse monoclonal (M2) as well as a chimera (rabbit). Myc The Myc tag is a 10 ...fluorescent marker. The GFP tag is ideal for live cell imaging, FRET, flow cytometry, or immunofluorescence where...

...would rather fund excellent people with mediocre ideas than mediocre people with excellent ideas. I am lucky...your life making connections and finding mentors -- well, it's time you call upon these people for guidance...Don’t go it alone.  You’ll find that no matter how well you know your idea, you will rarely succeed in launching...partners.  You need a dedicated team that you know well and trust.  This will allow you to discuss each ...two close friends, Sreyan Chowdhury and Clinton Buell, who have very diverse educational backgrounds (...

... Below you’ll find brief summaries of several excellent guest posts from our depositors. Interested in...Your Favorite Fluorescent Protein Oligomerizes in Cells GFP oligomerization can lead to misleading artefacts...which specific chemical inputs are received by a cell. These so-called recombinase-based state machines...required for viral replication and transcription as well as reporter genes. To learn how you can use minigenomes...used to deliver a genes of interest to a variety of cell types in-vivo. These are widely used by the optogenetics...activation and repression of different genes in a single cell, and localization of a fluorescent signal to your...

...community. Addgene's AAV preparations have worked well in my lab, including in applications where we have... can be used to deliver fluorescent proteins to cells as determined by the serotype. You can use these...determine if your AAVs are getting to the right cells using your particular delivery technique and conditions...expanding our viral vector educational resources as well, with both posts on the blog and pages on the Addgene...website you’ll find our updated viral vector pages as well as expanded virus protocols, information on how ...specific material page and includes western blots as well as other expression data. If you have any questions...

...Fhearraigh, PhD, of www.phdadvice.com. I sold out, well in part that’s what leaving academia feels like ...would continue on with my academic dream of studying cell division. However, after 6 years in the business...products, so you have some of the best ideas on how to sell these products to other scientists. Tailoring my...introduction to the financial world. It teaches you about selling products which is an important part of the business...Cambridge University where he studied mechanisms of cell division. Recently Seán got the entrepreneurial ...information on ELISA troubleshooting, ELISA kits and cell death assays. You can find Seán on twitter @Macfhearraigh...

...an awake monkey is different from a cell in a dish. I always tell people, 'Be driven by your science.'...for neuroscience back in 2005, both activators as well as silencers of neural activity. Since then, there...to Addgene in a matter of days. Addgene: Can you tell us a little more about this new two-color tool and...and see how different parts of the brain or the cells within them work together or perhaps compete against...two-photon properties, so you could activate single cells in arbitrary patterns in the brain. Addgene: Do ...

... between Cas9 (which causes the breaks) and host cell DNA repair factors (which repair those breaks) makes...example, correcting the allele that causes sickle cell anemia. However, HDR occurs much less frequently...a structure could drive sequence replacement in cells. Targeting three dCas9 molecules to a precisely ...functional Cas9 protein and the targeting guide RNA into cells by electroporation. Results from our lab and others... HDR. The detailed protocol for nucleofection as well as the sequences of our validated editing reagents...Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins...

...blogger Matteo Tardelli! Matteo Tardelli, PhD is Postdoctoral Scientist at Weill Cornell Medicine in NYC...was contributed by Matteo Tardelli, a postdoctoral scientist at Weill Cornell Medicine. Discussing data...Hamilton DP, Denfeld BA, de Eyto E, Hampton SE, Keller PS, Sharma S, Lewis ASL, Weyhenmeyer GA, O’Reilly...

...negative bacteria is due to differences in cell wall composition. Cell wall structure Gram positive bacteria...used in the staining process bleached another, Klebsiella pneumoniae. After realizing this was a repeatable...membrane. Common gram negative bacteria include salmonella and E. coli. Because of their thicker peptidoglycan...complex within the peptidoglycan layer, staining the cell purple. When the alcohol is added, it dehydrates...Silhavy, TJ., Kahne, D. & Walker S. The Bacterial Cell Envelope. Cold Spring Harb Perspect Biol. 2(5) a000414...

...different types of human cells, including T cells and hematopoietic stem and progenitor cells. Now with the DAP...to enable multiplex gene editing in a variety of cells and organisms. However, lengthy genetic payloads... over 50% averaging editing efficiency in human cells. MBE with DAP array also exhibited reduced Cas9-... editing, and nuclease editing, in virtually any cell type or organisms of interest with only minimal ...

...now.  Many thanks to our guest blogger, Kelly Hills! Kelly Hills is a founding bioethicist of the consulting...This post was contributed by Kelly Hills, founding bioethicist of Rogue Bioethics. On November 25, the... the MIT Technology Review dropped a bombshell report. A scientist working in China was using CRISPR/Cas9...separate things happened nearly simultaneously: The Wellcome Trust funded a center for mitochondrial research...consulting firm Rogue Bioethics. Kelly utilizes her firsthand experience in the software industry to inform...

...multispecies biofilm formed by Pseudomonas spp. and Klebsiella spp. Dr. Devarshi Gajjar, The M. S. University...research focuses on a collection of pathogenic Klebsiella and Pseudomonas isolates causing urinary tract...unknown regulatory regions in Pseudomonas and Klebsiella spp. The effects of these deletions on biofilm...major pathophysiological complications of diabetes mellitus. Recombinant human proteins that play a role in... Researcher who investigates the molecular and cellular etiopathomechanisms of ischemic stroke, the most... reduced blood flow. Novel treatment options, as well as non-pharmacological solutions to modify the course...

...of viruses used to deliver CRISPR components to cells or organisms under study. In the past we’ve left... they want to do is to quickly generate a single cell line and proceed to use it for experiments.” - ...screen for genes involved in a wide variety of cellular processes. While these are widely requested and...representation in the viral prep, infect target cells, and, finally, conduct the screen. To help alleviate...of this labor for our users, we now provide the Brunello (targeting human genes) and Brie (targeting mouse...deliver gRNA and therefore, must be used to infect a cell line already expressing Cas9. As John Doench (Broad...

... into the cell. This means that not all neurons and pathways will be transduced equally well by AAV-retrograde...they either trace downstream from the soma (neuron cell body) toward the axon (anterograde tracer) or upstream...them useful tracers, but finding a virus that works well for retrograde tracing can be difficult. The in ... Data Hub!) The cortical pathways to the IC were well transduced by AAVrg while other pathways such as...chemogenetic vector directly into the region where the cell bodies of the projection neurons reside.   Impact...

...electrocompetent cells in which the phage recombination machinery is induced using L-arabinose. These cells can be...function of certain regions in a promoter or gene, as well as to study the effects of inactivating/activating...coli to undergo nick repair. This method can work well, but poor template digestion can result in a high...required in recombineering. REPLACR-mutagenesis is also well suited for insertions and deletions. The figure ...Enzymes Required No No Yes Yes Lab Made Competent Cells Required Yes No No No Comparison of REPLACR-mutagenesis...

...stimulate AraC binding to I1 and I2 sites. Supplementing cell growth media with glucose decreases cAMP and represses...Steroid-regulated LexA promoter XVE (synthetic) pFZ19 (yellow flame) Temperature inducible promoters Temperature...gene transcription to take place. Addgene also has yellow flame light-regulated two component systems designed... Because transcription machinery differs between cell types or organisms, promoters are similarly variable...variable. Bacterial promoters only work in prokaryotic cells and typically only in the same or closely related... were derived. Similarly, the various eukaryotic cell types (mammalian, yeast, plants, etc.) require unique...

...government agencies, active researchers, as well as fellow iGEM teams. We were the proud hosts for the...industrial or research purposes requires stable host cells (the simplest example would be a transformed mesophilic...successful) as host systems since their biology is well studied, and they are convenient to maintain under..., since mesophilic chaperones would not function well under suboptimal conditions. This means recombinant...CRISPR-Cas mediated editing for psychrophilic systems as well as engineering and regulating psychrophilic recombinant...

...address all your unanswered questions via text! (well, maybe not all of them. We still haven’t gotten ...and would like to get it working in microglia as well. A:  We are not aware of any available AAV for efficiently...co-delivering different AAV particles into the same cells in vivo? A: This will be highly dependent on vector...particularly one that can target the epithelial cells in the blood-brain barrier?   A: For liver, we recommend...worth investigating for both Q6 & Q7.For epithelial cells of the blood brain barrier we would currently recommend...and to all our participants for their time and excellent questions.    Additional Resources on the Addgene...

...also gives cells antibiotic resistance to neomycin so that you can select the population of cells with the...transcription factor. The FLAG antibody has been well characterized and the system allows you to add this...Only a small percentage of the proteins inside a cell have a commercial antibody against them. There’s...antibody. It’s an epitope not found in human or other cells. It was made artificially. That means you aren’t...lab already should be able to run this protocol as well. We would also like to put some social pressure ...

...we virtually always use every single well in a 96-well (or 384-well) plate! How much do repositories help...in this post is a mathematical story well beyond my ability to tell. But even from these rough estimates...depositors have helped us save the scientific community well over $80 million on plasmids alone. (And we expect...

...product, the transformation may not result in any cells, or all the colonies screened might not contain ...i.e. primer3) or provided by the major companies selling PCR reagents. Nowadays commercial polymerases are...verify the strain and the efficacy of your competent cells (when they get old, they lose competency). If all... look at your experimental design. Full lawn of cells. The antibiotic in your plate might not work. Check... the incubator. The small colonies are called satellite colonies and they form when all the antibiotic...acknowledged in hundreds of publications including Cell, Nature, and other journals.   Paolo Colombi is ...

... be expressed in the same cell, but not necessarily at the same subcellular compartment as the opsin or...modulate the activity of neurons and other brain cells. Since the opsins or chemogenetic receptors used...neuropil conceals identification of opsin-expressing cells). For other things to keep in mind when making fluorescent...cerevisiae by use of an epitope addition method. Mol Cell Biol 8:2159–2165 . https://doi.org/10.1128/mcb.8.5.2159...Paré J, Bonaventura J, Richie CT, Michaelides M, Mueller SAL, Roseboom PH, Oler JA, Kalin NH, Hall RA, Smith...

...-HF1, & HypaCas9. The off-target problem It is well known that CRISPR/Cas9 genome editing can result... the EMX1 locus in human embryonic kidney (HEK) cells, 11 of these initial mutants retained the on-target...off-target activity at multiple genomic locations in HEK cells. Further testing of two of the mutants, SpCas9(K855A... showed that these mutants are not toxic to HEK cells and that similar mutations can improve the specificity... researchers to make precise edits in mammalian cells and may decrease worries about off target effects... Cas9 in complex with guide RNA and target DNA." Cell 156.5 (2014): 935-949. PubMed PMID: 24529477. PubMed...

...Communications, Harvard University Ray Howell -Founder of Howell Communications Philip Brenner, PhD - Assistant...scientific enterprise and the way it is funded have been well documented over the past 2 years (Alberts et al,... to shape the sector throughout their careers (McDowell et al, F1000Research, 2014; Dolan, F100Research...relationship, along with, of course, union dues. Time will tell the extent of the implications of this ruling and...27175853. Pubmed Central PMCID: PMC4866822. 3. McDowell, Gary S., et al. "Shaping the Future of Research...

...process will begin. Partnering with seqWell, we are using the plexWell technology to easily and quickly ...ever worked to sequence DNA on NGS platforms will tell you, while the process has become much more refined...assembly process. Again, thanks to our partners at seqWell we make use of a pipeline that takes the raw data...necessary adapter sequences for Illumina sequencing as well as unique barcodes per sample so we can assign sequencing...Tasissa M, Kostman M, Tillgren M, Makana Hanley L, Mueller I, Mitsopoulos A, Fan M (2020) A Novel Next-Generation...

...there isn’t a template strand left behind that the cell can use to repair. Figure 3: General overview...Occasional cleaving of the target strand is not as well understood but likely occurs in similar regions....complex in nucleotide unwinding steps. Molecular Cell, 70(3), 385-394.e3. https://doi.org/10.1016/j.molcel...formation by a type I-C CRISPR Cascade. Molecular Cell, 83(5), 746-758.e5. https://doi.org/10.1016/j.molcel...broad and unidirectional genome editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038...CRISPR–Cas systems for genome engineering in human cells. Nature Biotechnology, 37(12), 1471–1477. https:...