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...blot *indicates a diagnostic-only step Issue Potential Cause Troubleshooting Suggestion Proteins are...done the technical design of your blot, but are still finding yourself having issues, you’re in the right...type of gradient can help answer your particular question before running one.  Good on gradients? Excellent...the end of the post) has a number of helpful suggestions.  Gels Gel issues usually come down to voltage...want to try optimizing your transfer buffer.  Adjusting the buffer For wet and semi-dry transfers, you...membrane while increasing the ability of proteins to stick to the membrane by stripping away the SDS. To speed...blocking conditions on the spec sheet. If you’re still seeing high background after following manufacturer...

... “designer” channelrhodopsins with desired characteristics. The model was trained to predict if a channelrhodopsin...stronger photocurrents at low light intensity than existing channelrhodopsins CoChR and/or ChR2(H134R). Designer...however, have a higher conductance, so they can still depolarize neurons at lower MOIs. Figure ...located in the midbrain, fiber-optic cannulas were still stereotaxically implanted into the mice’s brains...reach the VTA. Mice were then allowed to “self-stimulate” their VTA dopaminergic neurons by triggering ...H134R) expressing mice did not have this self-stimulation response. This result showed that ChRger2 stronger... systemic delivery with PHP.eB compared to the existing ChR2(H134R).  In the second experiment, ChRger2...

...The Rare Genomics Institute BeHEARD (Helping Empower and Accelerate Research Discoveries) Challenge provides...our fourth year working with the Rare Genomics Institute by providing plasmid-based tools for rare disease...developmental delays or autism spectrum disorder. It is estimated that CTD is the second cause of male intellectual...psoriasis is a chronic autoinflammatory skin disease estimated to affect 0.27 to 4.6 per 10,000 people globally...-36 receptor inhibitors in development, there’s still a lot of mysteries surrounding IL-36 biology. “Little... the signalosome of different receptors upon stimulation. “To study cellular transduction events, we will...would allow us to address many of our research questions,” Hébert says. In the spirit of plasmid sharing...

...DEPC.  Ensure that plasticware or glassware used is RNase free. RNase-free plasticware is readily available...doing something without a kit has advantages: less plastic waste, less expense, and less of being left with...product to get rid of RNases such as RNaseZAP. When harvesting tissues, cells, plants, fungi, or bacteria, keep...extraction methods evolved into a simple protocol still used today There are many alternative methods for...-phenol-chloroform. In this method, samples are still homogenized and lysed in a guanidinium thiocyanate...hours, and variations on this no-kit method are still widely used today (Chomcynski and Sacchi, 2006)....Making the simple protocol even more foolproof (still without a kit!) As mentioned above, working with...

...contributed by guest blogger, Eugenia Rojas. A question worthy of a PhD: How do you visualize protein ...or in any other cell type. However, we cannot distinguish new from old proteins within that pool. Therefore...were quite tedious and, even here, there is the question of whether or not the tags influence behavior.... international PhD students at the Max Planck Institute for Biophysical Chemistry, Göttingen in Germany...in situ. I was in luck when I attended a very interesting talk by Prof. Erin M Schuman, a former Caltech...Caltech researcher, now Director at the Max Planck Institute for Brain Research in Frankfurt am Main, Germany...Germany. She presented a novel technology, back then still unpublished, that allowed the visualization of specific...

...Biotechnology from Worcester Polytechnic Institute where he investigated how plants achieve polarized cell growth... and Biotechnology from Worcester Polytechnic Institute. What is RNAi? The loss-of-function (LOF) experiment...any technique in the context of the biological question. For example, while gene knockouts have long been..., gene knockout strategies cannot be used to investigate essential genes. Historically, gene silencing...work has called this prevalent assumption into question. Specifically, CRISPR-generated mutants believed...present at the first stage of an RNAi-based investigation. Creation of the RNAi trigger requires a few...This process, known as “transitivity,” calls into question any claims about specificity of a given RNAi technique...

...Addgene Advisory Board member, and Institute Scientist at the Broad Institute, John Doench. CRISPR technology...nucleotide transitions but still requires a nearby PAM, although these are still early days for this technology...gRNAs to 5 – 65% of the protein coding region will still result in many dozens of gRNAs to choose from. With... While SpCas9, with a PAM preference of NGG, is still the most widely-used Cas enzyme, the development...basic landscape of mismatches that can nevertheless still lead to activity has been established, and can be... different sequences targeting the same gene, suggesting that off-target effects did not overwhelm true...the Genetic Perturbation Platform at the Broad Institute and has worked with many Addgenies to help improve...

...Drugs, DREADDs) and optogenetics as tools to investigate the roles of certain cell types in locomotion...themselves are not pathogenic, precautions should still be taken when using them. There are suspected cases...cause the least amount of immune response. Any investigator who is using an in vivo animal model should ... useful for a wide variety of experiments, care still needs to be taken when selecting which particular...do you choose the right serotype? Keep asking questions!  Table 1: AAV Serotypes and Tropism Experiment...transport, but they also have similar transport characteristics (velocities of movement, distance traveled,...excitatory connections (3). These facts beg the question: is axonal transport desirable or not desirable...

...and optogenetics meet, allowing scientists to investigate the fundamental basis of both brain function ...combined optical and genetic methods to photo-stimulate mammalian neurons and alter synaptic events: a...rhodopsins and neurons have in common? Neuronal stimulations are triggered by the movement of ions across...viral-infected neurons are subsequently photo-stimulated through fiberoptic cannulas directly implanted.... In particular, they found that optogenetic stimulations of the DA neurons produce behavioral and electroencephalography...could activate appetitive rather than aversive stimuli. In a recent paper, de Jong and colleagues used...used rAAV FLEx-based vectors to answer this question, mapping and characterizing the activity of the DA ...

...considerations for behavioral testing As a general consideration for behavioral testing in a laboratory, a high...where and when certain genes are expressed to investigate their role in a specific behavior. This blog ...influence on the outcome and cause variability in the testing group. For example, if an experimenter picks up...Mice may need to be given some time to adapt to a testing apparatus. General stress levels of the test animal...cleaned thoroughly between animals as olfactory stimuli that were left behind from a preceding animal (...behavioral neuroscience using viral vectors Behavioral testing is often done in conjunction with a genetic manipulation...behavior test. This time window between injection and testing will vary depending on whether it is an overexpression...

...other things, science communication comprises broadcasting, science writing, and certain aspects of art...2) Public engagement - Participate in science festivals and outreach If you are comfortable talking in...in public, volunteer at science festivals. Or engage with non-scientists in an unusual setting: Discuss...audience’s applause with your performance. When your institute opens its doors to the public, for example at ...your own blog, you can probably write for your institute’s blog. The Wellcome Trust blog or the John Innes...provides on-site courses in public engagement for institutions and the Royal Society gives training in media...citizen science project. Answering the big science questions around climate change, biodiversity, or genomes...

...of attendees were scientists still working at the bench. It was a fantastic opportunity to talk to so many... posited that “we underestimate living things.” He further cautioned the distinction of “higher” and “...Director of the National Human Genome Research Institute (NHGRI) of the NIH, started off with the first...unintended themes of the meeting was how much we still don’t know about RNA. One talk that really brought... gave a great introduction to the repeated and distinct aspects of the CRISPR regions of the prokaryotic... summed up this conference—nucleic acid science still has many surprises for the researchers in this field...

...pursue leadership opportunities at the local, institutional, regional, and national levels. These skills... But how does one gain such experience? Your institution may offer occasional workshops on leadership ...invaluable experience and even after 5 years, was still improving my skills. My involvement with the NPA...Harvard Medical School and then Boston University (institutions where I’ve worked as a postdoc). I soon realized...that many of the issues faced by postdocs and institutions are not local but are national issues. I applied...resulted in actionable tasks and I made a point of sticking to deadlines and performing the work I agreed ...every year it organized a “Committee Leadership Institute” (CLI) where each Committee Chair and Vice Chairs...

...techniques such as molecular cloning or molecular diagnostics.       Steps of PCR The beauty of PCR is...for this process as DNA polymerases require an existing strand of DNA to add nucleotides to.  Using these...The final PCR product is often visualized on a diagnostic agarose gel to confirm product presence, size...studying copy number variation, and molecular diagnostics.  Digital droplet PCR, ddPCR  Digital droplet...is determined and then analyzed using Poisson statistics to determine the concentration of the original... Cloning Site-directed mutagenesis Molecular Diagnostics qPCR Quantify starting template DNA concentration...number variations Quantify RNA levels Molecular Diagnostics NGS library prep QC ddPCR Determine ultrasensitive...

... as “instillation” or “gavage,” as they do not require any punctures. Intratracheal instillation Intratracheal...Skill required: advanced Intranasal instillation Intranasal instillation is very similar to its intratracheal... is best for you — and the mice. While not an exhaustive list, these can include what’s being injected...compounds, as circulation is slower, and more institutional oversight is required. Restraint: mechanical...Intratracheal instillation is used to deliver substances directly to the lungs. It is an advanced technique...gavage Oral gavage delivers drugs that require ingestion, such as experimental therapies or pain relief...

...There is no need for PCR or cloning, as digestion of the destination vector with SapI yields 2 sites- the...TEV-V5) with either blasticidin or puromycin selection. Researchers at the Allen Institute for Cell Science...sequences between the tag and homology arms). Digestion of the donor plasmids with SapI allows the tag... a tissue specific manner, as well as 3-site destination vectors for inserting a tag at multiple target...lines they’ve been used to create in the Allen Institute’s recent blog post. For more information on other...can be modified to assay additional protein characteristics. For example, Masato Kanemaki's lab has developed...

...Bioinformatics/HTS a journal club. Sorting out the logistics Once you decide on the topic, there are a number...journal club should aim to have many of the same characteristics as a successful meeting: a clear purpose and...different person responsible for each figure. Others still are more casual with a single presenter summarizing...open forum where people feel welcome to ask any questions they have.  These variations are suited for different...mix of closed (multiple choice/binary) and open questions is helpful to ensure acquisition of the essential...thoughts you may not have even considered. These questions should be tailored to the group and format but...but should almost universally include the questions “Would you attend this journal club again?” and “How...

...developed to break through the resolution barrier while still utilizing fluorescence microscopy, is another option...et al. 2016). That being said, the anchoring, digestion, and expansion steps can impact the integrity ...how well the gel polymerizes, and the level of digestion. All of these factors in turn depend upon the ... between them to ensure the expanded images are still a good representation of reality. Third, expansion...signal can be caused by loss of protein during digestion, or masking of epitopes during sample anchoring...steps. But even under ideal polymerization and digestion conditions, loss of signal often occurs, due to...secondaries, can improve signal strength (Sherry and Stiles, 2022). Fourth, signal continuity can appear reduced...

...analogous to traditional restriction enzyme cloning. Sticky-end mediated gene transfer would be particularly...off-target editing efficiency. Kim et al. and Kleinstiver et al. characterized genome-wide editing efficiency... to single mismatches, but variably so, with Kleinstiver et al. reporting that Cpf1 can tolerate mismatches...essential function in Cpf1-mediated editing, as Kleinstiver et al. saw no decrease in Cpf1 activity with ... are indicated below each graph. Figure from Kleinstiver et al., 2016. Cpf1’s strength may lie in ... off-target editing rates, determined using sophisticated genome-wide analysis. At many of its computationally...activity than that previously observed with SpCas9. Kleinstiver et al. suggest that AsCpf1’s off-target rate ...

...Description Gene/Insert PI Return to top Do you have suggestions for other plasmids that should be added to this...