...that can be easily maintained in a variety of biological systems. Read on for an in-depth breakdown of...insert and 1μg of plasmid backbone. It is also critical that as much of the backbone plasmid as possible...step, depending on the phosphatase you choose. CIP (calf alkaline phosphatase) or SAP (shrimp alkaline phosphatase...base pairs for each varies, it is difficult to calculate this based on DNA concentration alone. One method...varying ratios of recipient plasmid to insert. It is critical to set up a negative control ligation reaction...electro-competent cells instead of the more common chemically-competent cells. You should perform, at minimum...purifications, you generally lyse the bacteria; add chemicals to precipitate out the high molecular weight genomic...

...Deisseroth, K. (2005). Millisecond-timescale, genetically targeted optical control of neural activity. Nature...Ganguli, S., & Deisseroth, K. (2019). Cortical layer–specific critical dynamics triggering perception. Science... activated tools, can control everything from electrical activity to gene expression, and even CRISPR ...through multiple intra-cellular signaling cascades. Optical methods for controlling GPCR activation have been...

...expression by various mechanisms. The RNA guide is typically 20 nts and the genomic locus must often be proximal...promiscuous. In other words, they can sometimes localize to unintended sites with sequence similarity to... the on-target site. When a CRISPR-Cas system localizes to unintended sites and performs its programmed...product with CRISPR to (hopefully) be used in human clinical trials. An off-target rate of 5% is far too high...Cas-OFFinder, CCTop, and many more. While such software typically only considers DNA sequence and doesn’t take into...deadCas activator/repressor experiments where only localization of the Cas occurs. 2 gRNA approach Off-targets...effectiveness as a control. Since off-targets typically confound an experiment due to their location (...

...arguing with me. First I calmed the situation down by opening a dialogue specifically about the project and...We spoke about it for a while and he eventually calmed down, apologised and admitted he’d been under a...wherever it makes sense to start and try to be as chronological as possible — if you find yourself jumping backwards.... Your story should be too. Use drama Use any rhetorical technique you have in your arsenal to make sure...start point and it needs a good end point. One thematically pleasing option is to end at the present — ‘...

...Extremophiles - phylogenetically diverse microbes living in extreme physical and chemical conditions like...Stanford University became interested in exploring biological dark matter – large tracts of the microbial tree... to be diversity hotspots containing abundant biological dark matter and so the lab organized a sampling...like YNP thermal sites - are also of practical interest since they’ve previously yielded valuable enzymes...

...an antibody that works for your application, specifically detects your protein, is species compatible,...along with some pros and cons. FLAG FLAG is a synthetically derived 8 amino acid tag (DYKDDDDK) that can...possibly interfere with protein function and localization. If you need monoclonal GFP antibodies or guidance... N or C terminus of a protein may disrupt the biological function of some proteins. Protein folding issues... In rare cases, tags can also affect protein localization and solubility, so be on the lookout for aggregation...Overview of tag protein fusions: from molecular and biochemical fundamentals to commercial systems. Appl Microbiol...

...chromosome is present in this line, making it genetically female. HEKs are generally cultured as an adherent... cellular research tools have been optimized specifically for and in HEKs. These systems built around ...science research all the way up to the production of clinical viral therapies. Protein production  HEKs are...recombinant proteins at high levels. In a case where clinical applications of recombinant proteins are necessary...also grow in suspension, which allows for large scalability, and can also be adapted for serum-free growth..., which can make for a seamless transition of biological tools to other researchers using the cell line...

...conditions/locations but to engage the local community (including local garden and compost clubs) to participate...easy. Most of our equipment has been donated by local companies and BosLab has grown into an organization... for discovering novel therapeutics. Dave is a chemical biologist with a Ph.D. from UNC-Chapel Hill and...but are inspired to get involved, check out the local groups page on DIYBio.org to find a community lab...and follow us on twitter or facebook. If you are local, join us for our next event on meetup.com! Many...

...targeted cell ablation via ROS is achieved through localized expression of the FAP. A new photosensitizer:...the main advantages of MG-2I-dL5** over other genetically encoded photosensitizers is that it works with... ROS because NIR light doesn’t excite natural biological chromophores found in cells.  He et al also show...compared MG-2I-dL5** head-to-head with KillerRed, a genetically encoded red fluorescent photosensitizer protein...Watkins, S. C., . . . Bruchez, M. P. (2016). A genetically targetable near-infrared photosensitizer. Nature...Chepurnykh, T. V., . . . Lukyanov, K. A. (2005). A genetically encoded photosensitizer. Nature Biotechnology...

... from their original sources. Figure 1: Biological repositories that distribute mouse strains, plasmids... plasmids, cell lines, and much more have been critical for pandemic research. Image from Reproducibility...Cell Bank, part of the National Institutes of Biomedical Innovation, Health, and Nutrition in Japan. These... available to scientists around the world. This scale of production and distribution is possible due to...research, and mechanisms to maintain and grow these critical organizations should be a fundamental part of ... SARS-CoV-2 and SARS-CoV with implications for clinical manifestations, transmissibility, and laboratory...

... Social distancing? Many people are now calling this “physical distancing” since it’s important to stay...resources collated by Addgenies to help you keep calm and science on. Practice self-care and stay connected...in these is a great way to support your favorite local exercise spot or find something new. We usually ...message for some people. We should recommend #PhysicalDistancing. Social interactions and #social support ... COVID-19. You can also give back by supporting local businesses (ex: purchasing a gift card to use later...

...many opportunities available to those who are scientifically minded! Listen to Biotech Founder Michael Koeris...required Research skills The ability to think critically and carry out in-depth research is applicable...Scientific Advisor Lab Manager Patent Lawyer Technical Writer Meteorologist Journal Editor Textile ...organised, reliable, and can work independently. Practical outdoor skills are needed in a variety of roles...Breeder Scientific Illustrator Plant Breeder Zoological Worker Conservationist Documentary Director ...range of roles, including: Science Reporter Technical Writer Statistician Bioinformatician Science...

... transcription on the array have limited the scalability and efficiency of multiplex gene editing. Moreover... fused base editors (nCas9-BE), as Cas9 is mechanistically more favored for efficient base editing. They...been used for multiplex Cas9-nuclease editing. Typically, a single array encoding the tandemly assembled...released by endogenous tRNA processing machineries, specifically, RNaseZ and RNaseP. Although the Gao Lab demonstrated...loci MPE with similar high efficiencies, which is scalable for a higher number of MPE. Therapeutic potential...drive and process the tRNA-gRNA arrays, which is scalable and efficient in plasmid and viral vectors. In...

...algorithms are trained on vast amounts of data to calculate the relative immunogenicity score (Mattei et.al... evaluation of ‌therapeutic immunogenicity is typically performed using traditional 2D assays with immune...inform decisions about in vivo application and clinical testing. This would pave the way for improved ...in Macaca fascicularis: From Manufacturing to Preclinical Studies. In J. Bluemel, S. Korte, E. Schenck,...F. Weinbauer (Eds.), The Nonhuman Primate in Nonclinical Drug Development and Safety Assessment (pp. 519...2016). Antidrug Antibody Formation in Oncology: Clinical Relevance and Challenges. The oncologist, 21(10...

...Musicophilia includes case studies of relevant neurological atypicalities, including a patient with amusia - the...capture the attention of even the most skeptical reader of historical fiction (I can personally attest to...Science is a terrifying and equally good wake up call about the science of race."     - Joanne Kamens,...Strange & Mr. Norrell by Susanna Clarke "A quasi-historical/fantasy fiction. Two dueling magicians in the...

... Connie Cepko (Harvard Medical School), and Dr. George Church (Harvard Medical School) for taking the ...environment? Does the group expect each other to be physically present at certain times during the day? We asked...history in science. Potential graduate students typically go through a rotation, which reveals their working...job satisfaction (especially for scientists and technical people) is linked to engagement and access to ... "chaotic" and offer less micromanaging, they typically offer more flexibility in choosing direction of...

...such as plasmids) into mammalian cells, a process called transfection. Transfection is somewhat comparable... way. Selection methods, described below, are typically employed when creating stable cell lines and are...respectively). Therefore, it is not always mission critical to make sure the plasmid replicates in the cell...   ***The concentration used for selection is typically more (double) than that used for maintenance of...usually achieved by performing a "kill curve" (basically growing cells in various concentrations of the...resistance; however, like gentamicin, neomycin is typically used to suppress bacterial growth. The selection...

...specific subcellular localization peptide. By combining three different localization peptides with four ... for example).  Given that the liver is an immunologically active organ, this advance is likely to afford...off-target mutations. These plasmids contain catalytically inactive dCas9 and microbial SSAPs (dCas9-SSAP...system. dCas9-SSAP will make the editing of kilobase-scaled sequences more efficient and less risky.   ...plex mixed-subclone system using mStrawberry, localization peptides, and reporters that drive the expression...

...variables - your approach, your workflow, or your physical setup - to prevent the mistake, just like you ...differently.  Tools to Try  Checklists Found in surgical suites, air fields, and military bases, checklists...light-sensitive reagents” or “re-sterilize now.” Have a physical copy of the protocol when you work and make a ...time to do them carefully and thoroughly. Have a physical copy on hand as you run your experiments. Try ...your plate maps onto your plates, alongside your physical copy. If you’ve got messy handwriting, reach for...accuracy.  Bench setup Small adjustments in the physical organization of your workspace and your workflow...

...This process is called α-complementation. The system described above was put to practical use in the following...well-characterized bacterial lac operon contains a gene called lacZ that encodes for the enzyme β-galactosidase...β-galactosidase enzyme can break down a dye-linked substrate called x-gal (5-bromo-4-chloro-3-indolyl-β-D-galacto-pyranoside...deleting a section from the lacZ gene (a mutation called lacZΔM15) creates a non-functional β-galactosidase...Providing DNA encoding this section of amino acids (called the α-peptide) to a lacZΔM15-mutant bacterial cell...