...ligated into Cas9 or dCas9-containing destination vectors. Destination vector options include humanized wt...Addgene. 8. Xie, Kabin, Minkenberg, Bastian, & Yinong Yang. “Boosting CRISPR/Cas9 multiplex editing capability...editing. Multiplexing gRNAs: the basics One common question Addgene Senior Scientists receive is: can I express...ordered assembly of multiple components into a destination vector. The first step in CRISPR/Cas9 Golden ...oligonucleotides specifying each gRNA target sequence into distinct expression vectors using the enzyme BbsI. These...together to allow for ordered assembly into a destination vector that expresses Cas9. This is illustrated...drive ordered assembly into a Cas9-containing destination vector. Note: depending on which method you use...

... where you can type in questions for the speaker. “I think a lot more questions get answered this way ...audience can help decide which questions get answered. “You can upvote a question and the host will see the...upvoted question.”If the Q&A session ran out of time, the speaker could continue to answer questions in the...thought of walking up to the mic and asking your question in a large conference room puts butterflies in...the highly voted questions,” says Zhang. “Usually the moderator starts by asking the speaker the highly...audience engaged. They first sent a pre-workshop questionnaire to the participants to learn about what they... when you are giving a research talk, there are still other ways to make your session more engaging, for...

...prepares one for numerous non-academic careers. Still, many start their scientific careers hoping to follow... documentation of my results, answer loads of questions, and I also had to do some troubleshooting as ...ahead and contact a technical support team with a question or two that open a dialogue. This will give you...there is considerable diversity in the types of questions customers ask, and there are also differences ... in a technical support role. What general characteristics do jobs in technical support have? There are...Correspondent or similar. While there isn’t a clear distinction between these job titles, they may be different...team accepts any incoming requests and answers questions that are less time consuming and/or less technically...

...development of immunotherapeutics. There are still many important questions in this exciting field of research.... complicated and dynamic phenomenon that is interestingly responsible for tumor reduction as well as progression...inflammatory responses can augment tumor growth. Indeed, lasting responses to viral infections have resulted in ...chemical signals such as IL-8, IFNγ, and colony stimulating factor 1 (CSF-1). Additionally, tumor cells recruit...cell-surface proteome to inhibit expression of co-stimulatory molecules needed for T cell effector function...surfaces to avoid recognition by CD8+ T cells. Existing therapies and effects on immune system Conventional... However, these conventional therapies produce lasting side effects, damage healthy cells, and often cannot...

...startled by acoustic, visual or tactile stimuli, larval zebrafish react by performing a distinct C-bend to... first 2 weeks of life, larvae already exhibit distinct behaviors such as spontaneous swimming and escape...neuron more positive or more negative.  Neuronal stimulation: Depolarization When the inside of the neuron... NpHR, can even be expressed simultaneously and still be individually activated with different lights ...behavior is not done in response to any specific stimulus. While the escape response neural pathway is well...groups were activated or “turned on.” In order to distinguish between swimming behaviors, the researchers measured...immobilized in agar at the head with their tails still free, and different regions of the spinal cord were...

...techniques and is a multi-step process consisting of digestion, purification, ligation, and transformation...of the polymerase generates sticky ends in the PCR reactions. These sticky ends are annealed and ligated...used techniques in the biological sciences and constitute foundational elements of biomedical research....transformation. While restriction cloning is still routinely performed by many labs, a variety of other cloning... of cloning has its advantages, most scientists still encounter several struggles with these techniques...Restriction Enzymes + - PCR Amplification + + Digestion + + Gel Purification + - Ligation + - Transformation...restriction enzyme DpnI to remove the templates. Digestion products are then transformed directly into chemically...

...contributed by guest blogger Cameron MacPherson at the Institut Pasteur CRISPR software and the piñata effect ... group (Biology of Host-parasite Interactions, Institut Pasteur, Paris) that changed genome editing in...right software to use. With so much choice the question I have is, how did we get here in the first place..., like the miRNA bubble before it, offers an interesting view into how rapid innovation affects the research... lack of features in a single application. The question is then, should we use all tools as some kind ... can be used to aid design. These databases are still young and will require a collaborative effort from...arbitrary PAM definitions. These newer tools may still prove somewhat useful for the latest NTT targeting...

...Vincent Jean Pascoli, and Christian Lüscher. "Refining deep brain stimulation to emulate optogenetic treatment...Optogenetic stimulation of medial prefrontal cortex Drd1 neurons produces rapid and long-lasting antidepressant...norepinephrine to promote vigilance. A recent study investigated the injection of AAV8 particles expressing an...animal has been exposed to morphine or other stimulating drugs, it develops an enhanced propensity for... al., 2015) used optogenetics to support these existing findings by controlling neurons in the mPFC with...the open (risky). Taking advantage of these characteristic traits, rodents can be used to study anxiety....   A study from 2014 (Howerton at al. 2014) investigated the connection between neuroinflammation and...

...technique is still used today to test for HeLa contamination. HeLa contamination is still a problem today...Science (DBCLS),  National Cancer Institute, Public Domain, National Institutes of Health, Wikipedia user Aimecie...for a range of testing, from animal doping to forensics, DNA sequencing to drug testing. You can find ... there would be no viable viruses to study. Interestingly, due to certain high profile problems surrounding...favour of biochemical assays followed by in vivo testing. The mantra being “you can’t trust those whacky...provenance (this is the history of the cell line consisting of records of the origin and propagation of the...closely followed by T24 bladder cancer cells. Estimates of the amount of money wasted in basic oncological...

...resume for the job posting. For example, if the job asks for experience in “statistical analysis”, mention...mention “statistical analysis” (word by word) in your resume. If the job posting says “French-speaking is...Start browsing job postings and look at the jobs you are interested in. Consider listing the skills you already...their job postings and salary expectations public, so start by looking at these university postings to give...than 2 pages long. Write your resume for the job posting Don't think a resume is "one size fits all" like...handled large data sets and are familiar with statistical analysis. If you have done fluorescent imaging... yourself a sense of what to expect. If you are still debating whether to stay in academia or not, it ...

...science policy, public outreach, and restorative justice.  Follow her on twitter @sung_vanessa. Albany, ...students could walk around and ask scientists questions about their work. Thousands of people showed up...Science and Engineering at Rensselaer Polytechnic Institute. He also studies energy markets in his spare time...policies—many of which impact reproductive rights and justice. Whether it’s used to fight the myths that abortions...issues hit hardest. According to the Guttmacher Institute, most women of reproductive age live in a state...but we're now in a rift where our integrity is questioned and cuts to life-saving research are being proposed... us. I worried the march would be a simple, unquestioning celebration of science, at a time when scientists...

...recognized by the restriction enzyme MstII. When a restriction digest with MstII and southern blotting are conducted...knockout experiments to forensics and clinical diagnostics. One example of southern blotting being used ...are observed because the DNA has the site where MstII cuts. In a patient with sickle cell anemia the enzyme...real-time PCR to answer the same experimental questions. Real-time PCR detects and quantifies a gene or...saving the researcher both time and resources (3). Still, researchers generating transgenic animals may choose...commonplace, researchers used restriction mapping to investigate DNA sequence content by comparing the fragments...helped researchers at a time when sequencing was still in the early days of development. Today, researchers...

...eliminated by digestion/ligation and do not appear in the final construct. The destination vector contains...hands-on time, as digestion and ligation can be done in one 30-minute reaction. The destination vector and entry...IIS enzyme and ligase. Although the original destination vector + insert may spontaneously religate, this... to efficiently clone up to 7 gRNAs into one destination vector, making multiplexing easy.  Disadvantages...100% sequence-independent: to avoid undesired digestion, the Type IIS site used must not be present within...assemble. One way to work around this is to "domesticate" your fragment: PCR-based amplification can be...inactivation step. If your genes of interest or destination vector contain multiple internal restriction ...

...those questions! MTAs protect you and your institution, and almost all universities and institutions require...protective of both the providing institution and the receiving institution. Therefore, many technology transfer... when scientists are sharing their materials. Questions about intellectual property rights, pending publications...transfers. The UBMTA was drafted by the National Institutes of Health (NIH) and adopted by the Association...benefits for both the providing and receiving institutions and their respective researchers. Check out ...knowing your rights – and the rights of your institution – are protected while we handle all the paperwork... to you, or we can send it directly to your institution’s MTA coordinator. Organizations that have opted...

...at the Korea Advanced Institute of Science and Technology (KAIST) and Institute for Basic Science (IBS..., a researcher can cause them to interact by stimulation with blue light.  Similarly, light can also be... and is reversible upon termination of light stimulation. CRY2clust enables faster assembly and disassembly...integrated into a Ca2+ -modulating optogenetic system (OptoSTIM1), caused two-fold faster changes in intracellular...Listen to the soCoChR podcast segment Optogenetic stimulation is a powerful tool used by neuroscientists to...Finally, using synaptic receptor blockers to distinguish real and artifactual postsynaptic currents (PSCs...artifactual PSCs commonly observed with direct stimulation are largely absent in the soCoChR-expressing ...

...and called for the fast-tracked development of diagnostics. Mostly transmitted by Aedes mosquitoes (aegypti...regions of South, Central, and North America. Diagnostics will play an important role in helping to monitor...fellows with Jim Collins and Peng Yin at the Wyss Institute (Harvard University), we developed two new enabling...Pardee et al, 2014). As a demonstration of the diagnostic potential of these combined technologies, we ...of antibiotic resistance and strain-specific diagnostics for the Ebola virus. Our original proof-of-concept... the critical need for low-cost, field-based diagnostics, our approach could make an impact. Our motivation...paper-based platform into clinically relevant diagnostics for the Zika virus was daunting. It took a true...

... a summer role as a Research Scientist at the Institute of Cancer Research in Sutton, located on the edge...pediatric kidney cancer. This was particularly interesting as I had just completed my thesis on the study...tend to pay better. I worked in a Microbiology testing facility, where I tested materials for bacterial...bacterial contamination. It was a really interesting job at first, everything was very new, and everything was...job received many applications, employers were requesting that only scientists with an MSc or higher apply...to help cover the costs. My MSc courses were interesting and luckily genetics is a sector which has been...been rapidly growing. These factors left me optimistic that I would find a genetics related role soon ...

...seems captivated. Expertly navigate questions Answering questions is arguably the most difficult part ...Anticipate questions: When practicing with your colleagues, ask them to brainstorm potential questions you may...presentation veteran, but have difficulty answering questions. Or perhaps you’re simply attending, but uncertain...constructive criticism, and ask thought-provoking questions. For example, if you’re a virologist presenting...thinking about discussing a figure, jot down the question you were trying to answer, the method you used...your time: We often get so nervous when asked a question that we don’t fully hear or understand what we...have many times answered a completely different question than the intended one. Take a deep breath, and...

...cap: institutions of higher education nonprofit entities related to or affiliated with institutions of ...Having a lead role or critical role at a prestigious institution: This criterion is consistently under scrutiny...exempt from the cap if the applicant works at institutions of higher education, a related or affiliated...would not have been possible. Display of work in artistic exhibitions or showcases (not usually applicable...Proactivity is very important in this case.  You can still assist your PI in the peer review process, but remember...to self-petition. You need the support of your institute to do this one. Universities will usually only...professors. Casseus Law has seen that research institutions are willing to sponsor postdoctoral researchers...

... placing the dipstick into a wash solution removes contaminants while nucleic acids stick around. Finally...requirement for lab equipment. Plus they create plastic waste. Kits can also have the annoying tendency... bonds with water molecules and coaxes it into sticking to the hydrophobic glass filter. Each glass filter...commercial kits, but in less time. The authors estimate that a gravity-based column plasmid maxiprep kit...slurry that has a milk-like appearance. To make DNA stick to the glass particles in glass milk, gel slices...solubilizes both DNA and agarose, and 2) it helps DNA stick to the glass. After binding DNA, the glass particles...detergent, and ball bearings. Then a cellulose dipstick is dipped into this mixture to absorb the nucleic...