...methods. Since molecular, ecological, and reproductive confinement typically require very little effort...Development Fellow at the Wyss Institute and Harvard Medical School. Scientists making transgenic organisms ...the entire wild population with unpredictable ecological effects. Because the consequences of such a mistake... to implement and have been tested in yeast5. Ecological confinement involves performing experiments in...experiments attempting to build gene drives in tropical mosquito vectors of diseases such as malaria and...the laboratory. It varies by organism, but your local biosafety officer should be familiar with appropriate...they should not be relied on exclusively because historical studies of pathogen research have conclusively...

...club occurs with a small technical team (single lab or team), a deep technical dive on single papers could...number of participants and the standard academic calendar. So you want to start a journal club? Here are...topic, there are a number of administrative and practical decisions to make. How often will you hold it?... the time recurrently carved out. This notice typically should include the paper, any supplemental data...repositories, or other information needed by someone to critically discuss the paper.  Before the first journal ...primary purpose is training graduate students in critical reading. If the aim of the journal club is to ...Addgene, we opted to start with one per month. When to call it a day The most ineffective type of meeting is...

...used to calculate the concentration of the sample. This concentration can then be used to calculate the viral...viable lactic acid bacteria in faecal samples." Journal of microbiological methods 148 (2018): 64-73. PubMed... we have used quantitative PCR to evaluate the physical titer of our preps. We often repeat this assay...factor of 2. This means that if you set up two identical assays with the same sample, you could end up ...would be valid. This is why the AAV reference is critical. It allows you to determine if your titers are...adding your sample to a mastermix. At Addgene, we typically load 3 dilutions onto our ddPCR plate. As the ...Maintaining a high droplet count is important for calculating the concentration of your sample. Even if you...

...where you assume your technical skills define you and that the other not-so-technical skills you’ve built...industries and work environments. This is why they are called transferable skills. Why are transferable skills... individual with more going for you than your technical expertise in a specific field. While searching...under your belt from your last job. You knock the technical portion of the interview out of the park. It’s...hiring team will also want to examine not just technical skills, but also transferable skills, before offering... make hiring decisions. Though having all the technical skills certainly matters in the job market, it...you possessed. Think about your shining moments. Recall a few times you really felt great about your work...

...library! Now also available as a lentiviral prep. Calabrese: human CRISPRa sgRNA library In addition to inhibiting...lowly expressed genes or genes that are more drastically affected by gene activation rather than depletion... A and B. Sanson et al. demonstrated that the Calabrese library substantially identified more hits than...al., 2015). Root and Doench also compared the Calabrese library to screens performed with open reading...for resistance to MEK inhibition. Although both Calabrese and the ORF overexpression library identified ...in complement to study gene function. Find the Calabrese pooled library and lentiviral prep here! Applications...PMC4744125.  3. Gilbert, Luke A., et al. "Genome-scale CRISPR-mediated control of gene repression and activation...

... but this research is still in the pre-clinical/early clinical stages. Researchers are also working to... applications, and they’ve also had success in clinical gene therapy applications. Read on to learn more...gene therapy. In 2003, VIRxSYS began the first clinical trial with a lentiviral vector. CD4+ T-cells from...load and did not cause severe adverse reactions. Clinical benefits of lentiviral vectors have been observed...genome, they could promote oncogenesis by altering local gene expression. Self-inactivating (SIN) lentiviral...Patient monitoring and follow-up in lentiviral clinical trials. McGarrity GJ, Hoyah G, Winemiller A, Andre...gene therapy of human β-thalassaemia. Cavazzana-Calvo M, Payen E, Negre O, Wang G, Hehir K, Fusil F, Down...

...through mutagenesis in order to improve its physical and biochemical properties. In 1995, Roger Y. Tsien described...scientists to begin to study GFP and deduce its biochemical properties. Now GFP and its fluorescent derivatives...GFP is used in research across a vast array of biological disciplines and scientists employ GFP for a wide...tagging genes for elucidating their expression or localization profiles, acting as a biosensor or cell marker...living cells, enabling the study of dynamic, physiological processes. Re-engineering GFP to increase its...protein that undergoes conformational change. Typically, two fluorescent proteins with overlapping excitation...pH, using a range of strategies such as FRET, calmodulin, and others. Review Addgene's collection of fluorescent...

...analyzing your cells using flow cytometry, you are typically measuring the presence or absence of certain markers...free amines on and inside the cells and are thus typically used for distinguishing live from dead cells. ...atoms to a higher energy state. This process is called excitation. After a short period, the electrons...state, whereby a photon is emitted. This step is called emission. During excitation, some of the photon's... compared to APC. This happens due to a process called Fluorescence Resonance Energy Transfer (FRET). ...PE-CF594PE-Cy5PE-Cy7       Red (640 nm)   APCAlexa Fluor 647   Alexa Fluor 700APC-H7APC-Cy7 ...wavelength of 488 nm (Table 1), which is near a local maximum of PE's excitation spectrum (dotted line...

...Epitopes are between 5-8 amino acids long, with a typical length of 5 or 6 amino acids (Cruse et al., 2004... just as specific for their target antigen as canonical antibodies. They also have the advantage of being...recognize epitopes that full antibodies would not physically be able to reach. While a full antibody is ~150...of light that can pass through each well - the optical density (OD) - of the known and experimental wells...concentration of the known samples is used to calculate the concentration of protein in the experimental...surface of or inside whole cells. Antibodies - typically, primary antibodies that are conjugated to a fluorophore...the presence of specific proteins, but their localization within the cell or tissue as well. Similar to...

... a more-typical scale of tissue culture while still offering the potential for true biological insight...which to test the validity of your biological model and the technical performance of your assay. But, in...components, the model system and the assay are just as critical for success. Sometimes, you might have a few positive...involved. Or you don’t know how well your assay will scale. Or you don’t have enough cells to execute genome-wide...facilitating more rapid follow-up. We hope the biological community will find these reagents to be useful...

... prep library. Addgene’s pooled libraries are typically DNA preps, but we do distribute several of our...larger lentiviral preps can be used for screening-scale transduction of cells, resulting in cell population...The size and composition of your library can drastically impact what conditions it needs to properly amplify...can transduce your cells.  While Addgene uses a physical titer (via ddPCR) to quality control our viral...include a 1.3 mL aliquot with mostlibrary requests specifically to allow scientists to perform functional titering....  Resources and references References Genome-scale CRISPR-Cas9 knockout and transcriptional activation... useful place to ask experimental questions.  Practical Considerations for Using Pooled Lentiviral CRISPR...

...position at Addgene, the skeptical applicant asked if she would partake in “critical thinking” in the position... away from calling non-academic careers and desk jobs “alternative careers.” Realistically, based on the...it. The opportunity to be creative and think critically. One of the very first times I interviewed a ...wish for a stretch of time that doesn’t involve critical thinking. Former Addgenie Jessica Welch says, ...aspect of their chosen career. You can confidently call yourself a Scientist. You work in a lab. You formulate...transferable skills. Transferable skills are the non-technical skills you pick up wherever you work, and you’... communicating. After years of focusing on my technical skills with cloning and lentivirus production,...

... that could bind very specifically to target molecules. Using a technique called systematic evolution ... high specificity and affinity, were eventually called aptamers, from the Latin aptus, meaning “to fit...essentially mimicking GFP.  Though the aptamer, called Spinach, required longer exposure time than its...group selected for a 49-nucleotide aptamer they called Broccoli. As a product of SELEX and FACS, Broccoli..., Akhtar and Marty, Jean L. “Aptamer based electrochemical sensors for emerging environmental pollutants...PMCID: PMC4923196. 8. Sassolas, Audrey, et al. “Optical detection systems using immobilized aptamers.” ...9. Song, Kyung-Mi, et al. “Aptamers and Their Biological Applications.” Sensors (Basel). 2012; 12(1): ...

...institutions set their postdoctoral salary scales to the NRSA scale (Figure 21, National Postdoctoral Association...addition to certain staff scientists and those in technical roles. Therefore, institutions are responsible...suggested that the salary level should now be automatically adjusted every 3 years. In May 2016, Secretary...requirements of the FLSA. In the University of California system, the postdoc union has negotiated a raise...likely the number of postdoc positions in the biomedical enterprise in the U.S. will decrease. At Future...has clearly not been a sustainable approach to biomedical research in the U.S. and postdoctoral training...academic system is increasing, and becoming ever more critical. In 2016 not only were postdocs not exempted from...

...preference.   Historically, ICSS was done by implanting an electrode and non-specifically electrically stimulating...the steps and technical considerations are directly applicable to the rat [6]. Technical differences include...[2]. Laser activation is usually controlled automatically using motion capture software such as EthoVision..., regardless of the type of behavior, certain technical considerations still apply. Testing stimulation...is wrong or 2) there is some problem with the technical implementation of your optogenetics setup (viral.... 8.Han, Xue, et al. "A high-light sensitivity optical neural silencer: development and application to...

...signals from these reporters. Lack anatomical context/spatial localization: In vivo imaging with fluorescent... be overlaid onto anatomical ultrasound imaging to show where the bacteria localize in relation to internal...ultrasound fingerprint can be detected. Bourdeau et al calls this method acoustic collapse.  Importantly, acoustic...exciting application for ARG’s: in vivo imaging. Specifically, arg1 expressing E. coli aided in ultrasound...intestinal tract and has been used for 100 years to clinically treat enteric infection and inflammatory bowel..., but lux imaging shows only that the bacteria localize to somewhere in the mouse abdomen. arg1 signal...

...2012) scFv Antibody: Principles and Clinical Application. Clinical and Developmental Immunology 2012:1...being smaller than antibodies. scFvs are often genetically fused to other proteins, which has led to their...with 10-24 copies of the short epitope GCN4, also called a scaffold; and 2) a GCN4 binding scFv that’s fused...is easier and cheaper to generate since it’s genetically encoded.   Translational applications of scFvs...antibodies, there are many scFv-based therapies in clinical trials or already in the clinic (Bates and Power... tumour specific antigen. When these molecules, called Bispecific T-cell engagers (BiTE®s), bind CD3 on...David vs. Goliath: The Structure, Function, and Clinical Prospects of Antibody Fragments. Antibodies 8:...

...covered in detail here. Classical double-strand break repair (DSBR) In the classical DSBR pathway, the 3’...and Homology Directed Repair (HDR). NHEJ is the canonical homology-independent pathway as it involves the... provided the DNA template used for repair is identical to the original DNA sequence at the DSB, or it...plasmid, etc.), and consists of three pathways: classical double-strand break repair (DSBR), synthesis-dependent...purple arrows) or on the non-crossing strand (vertically at the orange arrows). If resolved dissimilarly...applicable in DSB-induced genome engineering, it has biological importance for the repair of breaks that lack...2281-308; (2013). PubMed. A Mouse Geneticist's practical guide to CRISPR applications. Singh P, et al. ...

...promoters and Shine-Delgarno sequences used in this so-called bicistronic design (BCD). The 49 vectors in this...: Čelešnik, Helena, et al. "Biosafety of biotechnologically important microalgae: intrinsic suicide switch... Rogers, Jameson K., and George M. Church. "Genetically encoded sensors enable real-time observation ... Ribosome-Controlled Fluorescent Reporter."ACS chemical biology 11.1 (2015): 31-37. pCri system The Gomis-Ruth...latency in matrix metalloproteinases." Journal of Biological Chemistry 290.8 (2015): 4728-4740. You can find...The gene set was carefully chosen to perform statistically relevant benchmarking of cell-free expression...Marx's lab has engineered and validated a new method called FREQ-Seq. This strategy allows scientists to construct...

..., soon directed himself to finding “immunological and virological solutions to crises like those and ended...Therapeutics, which was acquired by Ultragenyx Pharmaceutical in 2017. As one of the original employees at...learned more about Addgene. “The mission to share critical reagents such as plasmids and viral stocks for...depositor. KSQ uses a CRISPR-Cas9 platform to systematically test the function of all 20,000 genes in disease...Target Discovery at the Novartis Institutes for Biomedical Research. Drug development was something Stegmeier...genetics.” As a postdoc in Stephen Elledge at Harvard Medical School, he developed the first lentiviral microRNA-based...Associate Professor at Mass Eye and Ear and Harvard Medical School (HMS) where he leads a lab of 25 people....