...level, read on for more in-depth tips on probe chemistries, primer optimization, controls, and analysis....Picking your chemistry  The two most common qPCR chemistries are hydrolysis probes (which you probably know...commercial name: Sybr). However, there are other chemistries available and they can be the more optimal choices...choices at times. For example, chemistries that rely on hairpin design of probes/primers often offer higher...stable than the hairpin bond.  Alternatively, chemistries that combine the primer and probe into a single...comparison of features of several commonly used chemistries)   Chemistry Background noise Probe ...gel Table 1: Features of various qPCR chemistries. Adapted from Wong, 2018   Primers Design...

...studying the brain in a way that goes beyond such restrictions, you may want to consider a systemic capsid ...enhanced function after intravenous administration, distributing widely throughout the organism to target large...and recombination target sequences, denoted by triangles] facilitate tissue or cell type specificity. An...ultrasound (FUS), intracorporeal (IC), intracerebroventricular (ICV), intramuscular (IM), intrathecal ...your average AAVs — and have many of the same restrictions, like limited cargo space (~4.7 kb) for your...promoters and enhancers (Mich et al., 2021) can help restrict expression to your cell type of interest, while... with Cre-expressing transgenic mouse lines to restrict transgene expression to specific tissues and cell...

... a SmaI or XmaI restriction enzyme digest (the ITRs contain SmaI and XmaI restriction sites). Another ...these tools, I found myself writing AAV production tricks into the margins of my protocol. While these tips...recombination of duplicated regions like the ITRs. Restriction digest screening for ITR deletion is still necessary...ultrafugation Overview: Iodixanol gradient ultracentrifugation uses a gradient of different concentrations...during AAV production is overlaid. Following ultracentrifugation, the AAV-containing 40% fraction is collected...day. Refer to the AAV Iodixanol Gradient Ultracentrifugation Protocol for more details. Pro tips Watch...titer AAV. Do you have any AAV production tips or tricks? Please share them in the comments below! Special...

...This post was contributed by guest bloggers Becky Kucera, M.Sc. and Eric Cantor, Ph.D. from New England...hierarchical approaches using different Type IIS restriction enzyme specificities at each step. Factors such...limitations through the use of engineered Type IIS restriction enzymes and careful choice of junction sequences...involves the simultaneous activities of a Type IIS restriction enzyme such as BsaI, and a DNA ligase such as...receives the assembly after similar Type IIS restriction enzyme cutting results in 5’ four base overhangs... provide optimal reaction temps for both the restriction enzyme and ligase, thereby enhancing the ordered...destination construct and functioning Type IIS restriction enzyme and DNA ligase, any single omission should...

... This post was contributed by guest blogger Erik L. Snapp. Stop using EGFP/GFP for fusion proteins! Despite...inert. In all seriousness, EGFP/GFP has a real nontrivial propensity to noncovalently dimerize. That means... assumed that the molecules are homogenously distributed throughout the volume and can freely tumble in...be much higher than if they are homogenously distributed and mobile throughout the cell. If two copies...CFP, YFP, BFP, Cerulean, Turquoise, Venus, and Citrine) can be monomerized with an A206K mutation [2].... is sedimentation equilibrium analytical ultracentrifugation [2]. Some affinity assays used in the papers...protein concentrations. An imperfect solution is to restrict imaging analysis to cells expressing the lowest...

...research by banking and distributing monoclonal antibodies at cost. We distribute over 65,000 antibody units...The following post was contributed by the Developmental Studies Hybridoma Bank. Antibodies are the most..., is a repository for monoclonal antibodies, distributing over 3,000 antibodies and associated hybridoma...the University of Iowa. The minimal fees from distribution funds our operation. Antibodies are glycoproteins...PAX6, AP-2 alpha and ISLET-1. In addition, we distribute monoclonal antibodies against popular fluorescent...Posts about Scientific Sharing: The 10 Most Distributed Plasmid Technologies in Addgene's First 10 Years...

... media platforms are available or used in all countries; not all areas of any given country have equal...can guarantee you that either resource or time restrictions (or both!) will be a major limiting factor at...flexibility to visualize and contextualize. Can be distributed in digital or analog media formats. Cognitively...platforms can splinter audience. Primarily digital distribution (though radio is possible.) Written works...works Low Flexible. Low barrier to entry. Ample distribution channels both digitally and physically. Easy...overwhelming your audience. Digital or print distribution.  Static. Can be difficult to convey an entire...highlighting, or omitting details. Digital or print distribution.  Static. Often requires both artistic and scientific...

...This post was contributed by Patrick Miller-Rhodes, a Ruth L. Kirschstein NRSA Predoctoral Fellow at ...guest blogger Patrick Miller-Rhodes from the University of Rochester Medical Center. Patrick Miller-Rhodes...system (CNS). To address this technological gap, a trio of recent papers describe the development of CRISPR...method to dissect the individual and shared contributions of transcript isoforms to neuronal physiology...surface protein for which there are good flow cytometric antibodies, downstream of the TUJ1 coding sequence...these cell types are used. The lack of good flow cytometric antibodies for many CNS-resident cell types (...

...This post was contributed by Jim Woodgett. Public Library of Science (PLOS) created a stir earlier this... with limited acknowledgement of the initial contributors. This is probable to some extent - but that ...no one is suggesting all data be stored in a retrievable format, we too rarely preserve “raw” data of ... methodological detail. Publishers have also contributed to this issue by requiring ever more condensed...resources can offload the burden of data storage and distribution. I would argue that the journals themselves ...journal repository, they are likely too large to be retrieved by others. Other new data initiatives are also...and understand their data. Likewise, we want to contribute to helping others understand ours. Improving ...

... plasmids: Twitch sensors Griesbeck’s recent contribution to the field is a series of fluorescence resonance...which are available at Addgene, can be used for ratiometric in vivo imaging. Griesbeck explains, while these...both neuroscience and immunology. Don’t be shy: Ratiometric imaging for beginners Griesbeck says his calcium...beginners may have a tendency to shy away from ratiometric imaging, he recommends they give it a shot. “...practice optimizing the sensors involves plenty of trial and error. Transition metals also present challenges...dynamic range,” he says. Merkx recently applied this “trick” to the development of additional color variants...test.” References Thestrup et al. Optimized ratiometric calcium sensors for functional in vivo imaging...

... of times in 2 weeks. This is a striking example of the contributions that scientists are able to make... Figure 1: Biological repositories that distribute mouse strains, plasmids, cell lines, and much...Institutes of Biomedical Innovation, Health, and Nutrition in Japan. These results  will help researchers...investigation of this pathogen. The sharing and distribution of cell lines from repositories on opposite ...around the world. This scale of production and distribution is possible due to the dedicated facilities ...repositories’ capacity for storing, validating, and distributing such reagents are all ways in which the scientific...

...expert and the lack of credit for small contributions. We strive to eliminate these two barriers, creating...This post was contributed by Ignacio Amigo and María Hernández Sánchez from Crowdfight. In March 2020...between researcher and expert; 4) Credit all contributions.   Our volunteers are extremely diverse.... the project we are focusing on what we call “asymmetric collaborations.” These are transient partnerships...where a researcher makes a small but crucial contribution to someone else’s project. We think that these...A discussion about a protocol can save weeks of trial-and-error. A recommendation for the correct statistical...

...initiative since we started distributing plasmids in 2004. We’ve already distributed over 500 viral samples...In the middle of 2016 Addgene started distributing a small but growing catalog of ready-made AAV and ...the infrastructure capabilities to produce and distribute virus. Addgene is a 501(c)3 nonprofit and our...lead on operationalizing viral production and distribution, this project involved every Addgenie in the... to enable efficient, tracked production and distribution of viral samples. The Office Team made sure ... plasmid depositors and their institutions to distribute viral preps derived from plasmids in the repository...

...This post was contributed by Sreepadmanabh M, a member of the iGEM team from IISER Bhopal. Following ...The importance of psychrophilic enzymes From an industrial perspective, the enzymes obtained from psychrophiles...host The expression of recombinant proteins for industrial or research purposes requires stable host cells...either die out or grow so inefficiently that no industrial operation would ever consider them worth the ... Oleispira antarctica, and the P. haloplanktis Trigger Factor), that would allow the stable folding and...spirit with iGEM’s emphasis on holistic, community-centric science, our team has continuously engaged with...with a variety of stakeholders from industrial and commercial operators, government agencies, active researchers...

...useless, and excess people who are not contributing will detrimentally affect a team’s morale and efficiency...member-by-association of the Development Team, and I contribute to the Management Team that steers the organization..., when it came time to update how we make and distribute kits, I helped assemble a crack team of Addgenies...times have you ever seen a scientific article attributed to a single author? I’ve seen so few in my 20...to make progress towards open-ended goals whose metrics for success are defined by ongoing mission fulfillment... can get since participation on such a team is strictly voluntary. New members could be recruited, but... teams when I’ve realized that I really can’t contribute anything. Purging team members does not have ...

...This post was contributed by guest blogger, Kaustubh Kishor Jadhav, a Research Assistant at MGMs Institute...enhance the growth of mycoplasmas. The serum is nutrient-rich and also the best source for mycoplasma proliferation... longer time. When they come in contact with a nutrient source, they start proliferating shortly. Once...to the liquid or semi-solid medium (containing nutrients essential for mycoplasma growth). Infected cell...culture. Once these cells detect mycoplasmas, they trigger a series of chemical reactions and this induces...mycoplasma and then specific enzymes are used which trigger cellular mechanisms that generate luminescence....fluoroquinolone ciprofloxacin, ciprobay, zagam, baytril, tetracycline, etc. are available for mycoplasma...

...  AAV delivery of split base editors Article contributed by Jennifer Tsang AAVs are a great gene therapy...comparable to those currently used in human gene therapy trials. They further tested the base editors by correcting...genetically encoded voltage indicator Article contributed by Shreya Vedantam Genetically encoded voltage.... A MoClo toolkit for cyanobacteria Article contributed by Andrew Hempstead The Modular Cloning (MoClo... allows synthetic biologists to use Type IIS restriction sites to easily assemble multiple DNA fragments...applications including conversion of CO2 into water and electrical energy generation. Alistair McCormick’s lab recently...for precision in cell lineage tracing Article contributed by Erin Sanders CreLite, designed by the Eisenhoffer...

...the polyhistidine tag? The polyhistidine tag is a string of 6 or more histidine residues expressed in frame...using these more selective substrates along with stringent wash conditions.   Find polyhistidine tag plasmids...   Fig 2:  A, The polyhistidine tag is a string of histidine amino acids on the N- or C- terminus...tag placement can affect protein function. B, The string of histidines in the tag is typically between 6...normal protein function, however longer histidine strings can increase that risk.    Number of residues...of the tag, with longer tags you can use more stringent wash conditions and remove more contaminants without...antibody, like this one developed by Dr. James Trimmer’s lab, to perform assays that they otherwise would...

... optogenetics toolbox with CRY2clust Article contributed by Brook Pyhtila  Listen to the CRY2clust podcast...dynamics, higher sensitivity to light, and better distribution of clusters without accumulation in subcellular...that enables single-cell optogenetics Article contributed by Meghan Rego  Listen to the soCoChR podcast...        Synthetic decaffeination operon Article contributed by Angela Kaczmarczyk  Listen to the synthetic...enables the degradation of caffeine (1,3,7-trimethylxanthine) and its conversion into xanthine. The majority...live-cell imaging of biochemical activity Article contributed by Angela Holmes Jin Zhang’s lab is developing...the FLIM-FRET acceptor podcast segment Article contributed by Christina Mork The Murakoshi lab has developed...

...added to the wells. This reaction produces a colorimetric reaction that reflects the concentration of ... concentration or antibody specificity via a colorimetric reaction. Image from Boguszewska et al., 2019... concentration or antibody specificity via a colorimetric reaction. Image from Boguszewska et al., 2019...added and the reaction can be visualized via a colorimetric reaction. The direct ELISA is often used when...concentration of proteins in an ELISA well is based on a colorimetric reaction. Most commonly, this is a blue reaction...