...cells; for each cell type, the transfection/delivery step needs to be optimized to achieve high knock-...IVT sgRNA) approach (Dewari et al. 2018 and Figure 2). Not only is the cr:tracrRNA approach highly efficient...biochemical tasks in the cell. One of the most popular approaches to study protein interactions is the pull-down... generate in-house) a pre-validated antibody for each target protein. To study 20,000 human proteins, ...throughput because numerous cell lines can be generated, each with a different tagged protein of interest, and...Using our 96-format pipeline, we can routinely achieve 5-30% tag knock-in efficiency across different ...experiments. You’ll need to optimize csRNP delivery for each cell type you work with (optimization includes selection...

...while minimizing confounding secondary effects? An approach described as “knocksideways” (a British idiom ...Gateway cloning methods developed by the lab of Joachim Wittbrodt might be just the set of tools that you...construct. Up to eight entry vectors can be used for each Multisite Gateway compatible destination vector ...protein from expression to degradation. Chemical approaches may require expensive peptides or small molecules...and Technology Austria, decided to take a novel approach to control for these effects, drawing on the rapidly...optogenetics. Using a rational protein engineering approach, they designed Opto-RTKs, which activate signaling... on exposure to low-intensity blue light. This approach relies on the incorporation of light-oxygen-voltage...

...classical TANGO assay, Kroeze et al. developed a new approach termed Parallel Receptor-ome Expression and Screening...to test multiple expression systems before they achieve the appropriate expression level for their gene...signaling pathway and includes 180 unique transcripts. Each gene was chosen to represent the primary splice ...fly genes with at least three independent gRNAs, each chosen strategically to maximize the likelihood ...generated 5 common 30bp overlap sequences (Sites #1-5). Each overlap site encodes a unique restriction enzyme...limited, as prior optimizations are required for each individual target species. Indeed, in order to use...system for targeted DNA methylation The traditional approach to studying epigenetic modifications has been ...

...either “shock and kill” or viral excision approaches, and each of these gRNAs will need to be validated...of the integrated HIV-1 genome. For a targeted approach, Zhang et al. turned to the CRISPR/Cas9 Synergistic... essential to HIV-1 replication. However, this approach could permit some viral protein production, so...animal models, and it’s difficult to tell if one approach might be more successful. “Shock and kill” has...HIV-1 to deplete the viral reservoir, and this approach doesn’t require a functional Cas9 nuclease, so...hurdles. The first is the delivery of the CRISPR machinery to all of the target cells. This goal may prove... reservoir spans multiple organ systems. Both approaches may prove more successful early in the course...

...conference room puts butterflies in your stomach, the virtual approach might be less stressful. For both conferences...conferences have followed suit. Two Addgenies, Susanna Bachle and Yijun Zhang, recently attended virtual conferences...walk 20 minutes between one session to another,” Bachle says. “Now, the physical location didn’t matter... asking in front of many people is lower,” says Bachle. (For presenters, there’s also less stage fright...Meetup, hosted their poster session on Twitter where each poster presenter had four “slides” to use for their... meeting, I received a lot of LinkedIn invites.”Bachle pointed out that networking at virtual conferences... to take more initiative. “You have to actively reach out. It’s more proactive versus organic networking...

...can we make a decision for each patient if the majority of information on each cancer clinical sequencing...present, and use prior knowledge (data connecting each genotype with vulnerabilities) to design a therapeutic...and most patients still don’t benefit from this approach. One central bottleneck is that most recurrently...computational algorithms, such as SIFT or PolyPhen to approach this challenge, using attributes of cancer variants... would be extremely helpful as a complementary approach. Also, in principle, these experiments could find... designed to complement the one assay per gene approach that has been the historical standard. Specifically...team have recently demonstrated the power of this approach: Phenotypic Characterization of a Comprehensive...

...DNA-binding proteins to label certain loci, but this approach is not scalable for every locus...unlike CRISPR...distances from 2-75 Mbp. Labeling multiple, intrachromosomal loci Building on the work of Chen et al., ... to SpCas9 and its orthologs NmCas9 and St1Cas9. Each ortholog was fused to a different fluorescent protein...The specificity of these orthologs is key: since each ortholog requires a different PAM sequence, a gRNA...turned their attention to mapping pairs of intrachromosomal loci. The technique successfully resolved ... this work represents the first mapping of intrachromosomal loci, a major benchmark in characterizing ...-MS2-PP7 Despite the success of Ma et al.’s approach, fluorescently labeled Cas9’s come with a number...

...their success. But how do you find these elusive teachers, supporters and advisors? I tried to start a more...’s Association (HBA) Boston Chapter and the Massachusetts Association for Women in Science (AWIS) mentoring... enough, especially in underrepresented groups) reach multiple mentees efficiently Allows all group members...development stage, they have a lot to learn from each other. Some have taken my advice and I recently ... you want to cover during the year. Suggest that each person bring 3 topics and write them on a board ... minute lab emergencies are no excuse).  Assign each meeting a leader from the group in a rotation. The...regular meetings. Experiment with the process. Have each person write a Development Plan. Set concrete goals...

...Targeting and manipulating interneurons using a viral approach Genetically modified organisms like transgenic...successfully targeted via viral particles, an effective approach to target specific neuronal subtypes was not available..., Gordon Fishell’s lab has developed a new AAV approach using regulatory elements that restrict expression...tools in virtually any vertebrate. This novel AAV approach allows you to target and functionally manipulate... out our recent guest blog series: A Practical Approach to Choosing the B(right)est Fluorescent Protein...With eSpCas9(1.1) and coselection, Agudelo et al. achieved editing rates as high as 83% with NHEJ and 40-... of available target sites for base editing. For each base editor, they observed editing activity with...

... Targeted sequencing approaches More targeted alternative sequencing approaches have been developed. ...lower them even further. While not all of these approaches will work for every experiment, at least one ...where only localization of the Cas occurs. 2 gRNA approach Off-targets that come from mutations arise from... Cas nickases. When two nicks are generated near each other, this will result in a DNA double strand break...off-target sites within a close enough distance to each other to cause a DSBs will be very low, reducing...especially when DNA mismatches are involved. Two other approaches have been used - GUIDE-Seq and integrase-defective...site-specific detection downstream. The con to these approaches is that NHEJ must perform the repair to allow...

...for each primary antibody that will be used (Burry 2011). It does not need to be repeated for each experimental...of biological replicates such as multiple mice in each group and C, technical replicates or repeated measurements... unavailable, try substituting with knockdown approaches using siRNA or shRNA. If this alternative is ... alternative, albeit less ideal, verification approaches discussed in the Antibodies 101: Validation post...multiple primary antibodies, include controls where each primary is omitted but all secondaries included ...Secondary antibody specificity  In indirect staining approaches, secondary antibody specificity must also be ...secondary antibody control should be included with each experimental run (Burry 2011). Autofluorescence ...

...secondary milestones to meet along the way to achieving each aim. A funded proposal typically lists the ...opportunities to convince others why a project is the best approach to a given problem, Empowers the writer, And... for example, and tips to consider while writing each section. Research challenge The Research Challenge...While some of the statements may be slightly far reaching, remember that you are writing a proposal to test...use it to confirm that your solution is the best approach to the research challenge. Demonstrating expertise...Specific aims The Specific Aims section highlights each milestone that must be met in order to deem your...be challenging to master at first. Additionally, reach out to peers and collaborators who have succeeded...

...copies of VP64 at each loci of interest, this allows more transcriptional machinery to be recruited per...TALE proteins, proteins had to be re-engineered for each gene, making wide-scale gene activation seem next...activation: rather than requiring protein engineering for each loci, CRISPR/Cas systems only require changing the...Guided by dCas9, VP64 recruits transcriptional machinery to specific sequences, causing targeted gene regulation...levels. Although other methods have been able to achieve much higher activation, dCas9-VP64 is great for...Description Rather than using a single copy of VP64 per each dCas9, SunTag uses a repeating peptide array to ...JL, Davidsohn N, Housden BE, Perrimon N, Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient...

...that you come into the lab wishing there was a machine that could make your life a hundred times easier...off our hands. Good news! There is probably a machine or robot out there that does exactly what you want...lot of time. Enter the Biomek FXP! We wanted a machine that would improve our transformation protocol ...played a factor in our decision to go with this machine, but we also really liked that Beckman was flexible...apart before plating. With this fine piece of machinery we have been able to transform 96 samples in about...samples at the same time as opposed to preparing each sample individually. Enter the BIOROBOT! This robot...lab team. Enter the Liquid Handler!     This machine fits on a table top and can be easily programmed...

... the fluorescence amplitude measured for each droplet in each well (Figure 2). A clean ddPCR should have...a fresh one for each set up.  Wipe down the bench and all consumables with 10% bleach.  Pipette slowly...20,000 droplets using water-oil emulsion technology. Each droplet contains the ingredients for amplification...dilutions onto our ddPCR plate. As the titer is unknown, each dilution should fall within the range of the assay...emulsion containing approximately 20,000 droplets. Each droplet contains the material required for a mini...plate and measures the fluorescence amplitude of each one. Droplets that fluoresce contain the amplified...is common. Here are some steps you can take to achieve a clean NTC: Have a dedicated bench with a dedicated...

...Ig) heavy chains and two Ig light chains make up each antibody molecule. The Fc region and part of the...IgG, or IgE). In the context of the immune system, each isotype is expressed at different times of the immune...system production and many more engineered methods. Each type of antibody and production strategy has advantages...advantages and disadvantages compared to each other. Polyclonal antibodies These antibodies are derived...recognize multiple epitopes on the protein. However, each animal will make antibodies against different protein...full antibodies would not physically be able to reach. While a full antibody is ~150 kDa, a nanobody is...similar concentration of cell lysate was added to each well. Image from Sun et al., 2010.   Enzyme-linked...

...adeno-associated virus (AAV), from reaching their targets. The AAV9 serotype can breach the BBB following injection...but requires a high vector dose and is unable to reach all regions of the CNS. PHP.B, a neurotropic variant...C57BL/6J mice following IV injection but fails to reach the CNS of BALB/cJ mice. These results were surprising...C57BL/6J mice strains to identify variants that reached the CNS following IV injection. Compared to AAV9...antigen-1 (SCA-1), is the receptor used by PHP.B to reach the CNS; yet LY6A is an unlikely candidate. It was...transduced by PHP.B. The Deverman Lab took a broad approach and searched for genetic difference between the...phenotype in mice. They took a classic genetics approach and interbred C57BL/6J and BALB/cJ mice to map...

...presence of that metabolite (Fig.2). Spinach eventually evolved into Spinach2, a more stable and brighter version...leading to a positive signal. The same effect can be achieved for a fluorophore that relies on dimerization ...lack of fluorescence for readout may render this approach less sensitive in most applications. Learn About...cleavage and an RNA aptamer that bound the compound malachite green (MG) to create a non-protein based visualization...essentially mimicking GFP.  Though the aptamer, called Spinach, required longer exposure time than its protein...possibilities. For instance, further selection of Spinach with a target metabolite creates an aptamer that...also has a higher melting temperature than its Spinach ancestors, and has a lower magnesium dependence...

... express a gene of interest. While the general approach for using lentivirus, infect and select, seems...mycoplasma can be extremely hard to detect with levels reaching 108 cells per ml before the media becomes cloudy... cell lines, ELISA, fluorometry, or PCR-based approaches to detect even low levels of contamination. For...Uphoff et. al. outline a do it yourself PCR-based approach complete with freely available positive controls...benefit of the multiple harvest strategy is that each day you are supplying your producer cells with fresh...envelope. One drawback of the multiple harvest approach is that it tends to result in a higher volume ... your lentiviral prep!) and infect. While this approach is a great start to a successful infection, one...

... was weird like that,” says Caitlin. A love of teaching also runs through Caitlin’s blood. “I come from...faculty members, I helped grad students who were teaching for the first time. I walk[ed them] through their...them advice, [and helped] proctor exams.” These teaching experiences led Caitlin back to being a student... signed up for a 10 week class at Iowa called “Teaching Your Research.” This course has participants take...you know about it [your area of research], and teaches you how to design a course, how to design good ...ground up.”  Science writing and editing These teaching experiences definitely weighed in Caitlin’s post-grad...but in the end, writing won out. “I considered teaching at a liberal arts school but I’ve always enjoyed...