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...are not general amplification protocols. Rather, each protocol is optimized for a single, specific library...step also depends on the individual library. So, on each library’s page, you can find protocols for sequencing...protocol on Lentivirus Production provides a general approach for producing lentivirus in Lenti-X 293T cells...virus–the right amount of virus, that is. Ideally, each cell will be transduced by no more than one viral...

...required to develop new projects and test novel approaches, a necessary skill set for any scientist. In ...lab   Thinking Outside the Box: Individual Approaches Whether it’s finding the source of contamination...your situation.   Thinking Outside the Box: Team Approaches At some point or another, you’ll find yourself...benefits from their policy! Perfectionism When we reach for perfection, our work stalls. We become so fixated...

... work my lab wanted to do already seemed to know each other and what to do.  Saved by the Tweet - The...people attending large meetings. A key was to provide each bin with a mentor who had attended the meeting multiple...networking and mentoring circles We looked at a lot of approaches scientific meetings have taken to provide mentoring...Joe taught high school chemistry for a year. The teaching/mentoring bug never went away. While he has frequent...

...primers for each individual sample, this will reduce convenience and comparability because each sample with...other DNA quantification methods - see below), since each viral particle typically contains one viral genome...indication of infectious titer, consider titrating each lot of AAV in vivo to determine the optimal dosage...

...rewarding parts of the job. Solutions may also be achieved by simply replacing the product in question. This...different backgrounds and realizing that the same approach does not work for everyone. The benefit is that... in project management, consulting, scientific outreach, and more. Technical support roles also involve... images used in this post. Klaus Wanisch is an Outreach Scientist at Addgene. These days he's doing a ...

...are many types of electronic lab notebooks (ELNs), each with its own pros and cons. All ELNs have the virtue... take a look at the RSpace ELN.  The connected approach in action: how the RSpace ELN supports reproducibility...research, and data repositories. Below I describe how each of these kinds of connectivity facilitates reproducibility...electronic lab notebook. Rory is based in Cambridge, Massachusetts, and feels privileged to be part of the vibrant...

...recombinase have been developed. A common means to achieve ligand control is to fuse a mutant estrogen receptor...be transfected by in utero electroporation. To achieve sparse labeling of individual neurons one could...the same time from two different plasmids this approach becomes problematic as it reduces the probability... the Saccharomyces Cerevisiae 2 Micron Plasmid Attaches Covalently to DNA via a Phosphotyrosyl Linkage...

...salaries). Postdocs (who are not in a primarily teaching role) come under this ruling, regardless of visa...on higher education, you can see that those in teaching roles (such as adjuncts) and graduate and undergraduate...policies. It is unclear how many postdocs are at each of these institutions (populations potentially from...trainee workforce has clearly not been a sustainable approach to biomedical research in the U.S. and postdoctoral...

...and longer genome search times. Using a machine learning approach, the lab proposed multiple sets of Cas9...editing was deemed to pose low risk to KJ. This approach won’t be acceptable in every case, however, and...CRISPR-Cas9 PAM variants via scalable engineering and machine learning. Nature, 643(8071), 539–550. https://doi.org...

...therapeutic settings. Silencing of a target gene is achieved through the delivery of a double-stranded RNA ... plasmids, shRNAs hijack the endogenous miRNA machinery for processing (Figure 1). The shRNA sequence ...the bound miRISC can simply block translation machinery. The composition of the RISC varies, but at the... Silencing Complex: a versatile gene-silencing machine. Journal of Biological Chemistry, 284(27), 17897...

...tag. Many thanks to our guest blogger, Joachim Goedhart! Joachim Goedhart is an assistant professor at ...This post was contributed by guest blogger Joachim Goedart, an assistant professor at the Section of ...fluorescent probes. You can follow him on twitter: @joachimgoedhart.   References Adjobo-Hermans, Merel JW, et ...

...desirable traits using traditional plant breeding approaches, these techniques are cumbersome, often requiring...highlight the specific modifications to CRISPR machinery that allow for the use of CRISPR in plants, and...in question. In plant cells, gRNA expression is achieved by placing the gRNA downstream of a plant-specific...Glycol (PEG), although the application of this approach is limited to protoplast cells (plant cells whose...

...allowing for the study of their function. This approach to studying DNA sequences helped researchers at...Researchers perform four different reactions, one for each radiolabelled ddNTP, and then run these reactions...generally considered first generation DNA sequencing machines (9). Today, similar technologies, sometimes referred...older technologies nanopore-based DNA sequencing machines are relatively small meaning that DNA sequencing...

...small number of glutamate molecules are released in each synaptic event and they are rapidly taken up by ...for two-photon (2P) imaging and loose-seal, cell-attached recordings in primary visual cortex (V1) layer... is scarless, unlike previous integrase-based approaches. Find STITCHR plasmids here! Fell, C. W., Villiger...'s important to know what proteins interact with each other and where those interactions occur in the ...

...homology on each side. For optimal repair, place the loxP sites precisely at the cut site for each guide. ...When delivering the ssDNA template and CRISPR machinery, Quadros et al. recommend ribonucleoprotein (RNP...strands, as well as guides facing towards or away from each other.  Figure 4: How to design a conditional...

...role model Make sure the mentors you choose are teaching the stuff you want to know and display character...members speak disrespectfully of supervisors and/or each other. You are not allowed to speak to lab members... a mentor that sees you only as a paper making machine. Choose one that is a true mentor, supporting career...collaboration? If you are considering a career at a small, teaching college you might want to consider less expensive...

...it is also a good idea to run an uncut sample of each plasmid to help with troubleshooting if your digests...approximately 1:3. Since the number of base pairs for each varies, it is difficult to calculate this based ... alone. One method is to conduct 2 ligations for each plasmid you are trying to create, with varying ratios...2), or roughly equivalent numbers of colonies on each plate (unsuccessful 3).     If you have a high ...

...many times, until you achieve whatever goal you’re aiming for. When you complete each cycle, your best-performing...building things, lots of math, and maybe heavy machinery. But not all engineering is at a large scale —...improved specificity. There are different ways to achieve this goal, including error-prone PCR, degenerate...

...blog post will discuss differences between these approaches, and provide updates on how best to design gRNAs...should not be undertaken lightly! Indeed, truly achieving the gold standard requires not one but two rounds... using knockout or transcriptional modulation approaches. Avoiding off-target effects The off-target ...Guell M, Byrne S, Yang JL, De Los Angeles A, Mali P, Aach J, Kim-Kiselak C, Briggs AW, Rios X, Huang P-Y, ...

...to arrive at this hierarchy, the main aspects of each class and type are easy to understand. Classes are...involved in plasmid competition or can hijack machinery from other CRISPR systems to carry out various...further broken down into type II, type V, and type VI, each relying on a unique Cas effector — Cas9, Cas12, ...basic diversity and the surface-level function of each type could lead you to new directions in your research...