...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...

...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...

...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 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...

...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 ...

...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...

...  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...

... 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...

... by ultracentrifugation (75,000 - 225,000 g for 1.5 – 4 hours at 4 °C). After ultracentrifugation, virus...integrity of your virus, perform a diagnostic restriction enzyme digest and compare it to some reliable...vectors) you can concentrate your virus by ultracentrifugation of the viral stock, followed by resuspension...packaging cell debris before proceeding to ultracentrifugation to avoid contaminating your transduction ...through a 0.45 µm or 0.22 µm filter) or by a centrifugation step (5 minutes at 300-500 g). After removing... to concentrate a virus solution without ultracentrifugation, you can reduce the volume of cell culture...

...and sensing electrical potential. Thankfully, there is a tool to measure these electrical events! Voltage...biological tools. Why Use Voltage Indicators? Electrical signals are integral to many facets of biology...muscle contraction – is directly related to the electrical impulse of the cardiac cells. To study this phenomenon...is carried out by action potentials which are electrical charges propagated through multiple cells to ...depends on the ability to monitor their signals – electricity! The research tools for studying voltage within...was dipicrylamine, a lipophilic compound that distributes itself within the plasma membrane as well. Another...

...days Purification Ultracentrifugation (AAV); centrifugation (lenti) Centrifugation Titration time ~...using methods like ultracentrifugation, while lentivirus supernatants are centrifuged and filtered to eliminate...have a higher titer than a P1 virus. Pro-tip! Centrifuge the viral supernatant in between passages to ...straightforward — simply spin down the supernatant in a centrifuge to remove any unwanted cellular debris, as you...

...This post was contributed by Jake Watson and Javier García-Nafría from the MRC Laboratory of Molecular...present even in common lab cloning strains. This intrinsic bacterial recombination pathway, referred to as... longer oligos, which could be avoided using restriction enzyme cloning, were more prohibitive at the ... doesn’t matter if the DNA is PCR amplified, restriction digested or even a synthesized gene. PCR is the... Mutagenesis is performed by designing primers astride the old codon, amplifying the whole vector. The...extending homologous sequences (to 25-30 bp). Tips and tricks for IVA cloning Use a sequence manager A sequence...IVA can be employed by cutting the vector with restriction enzymes and adding the corresponding homologous...

...colonies in petri dishes, but close to 80% of samples are received as DNA in a microcentrifuge tube. Once...sample enters Addgene’s lab, a series of events is triggered. Each step is tracked through barcoded tubes that...track all of our 40,000 samples available for distribution!    Check Out How We Think about Developing...The preferred E. coli strains for storage and distribution at Addgene are DH5α, NEB stable, ccdb survival...Each sample that is transformed is plated onto a petri dish plate that is also barcoded. The barcodes on... to be transformed with a micropulser using an electric charge to help the DNA enter the cells. Some samples...

...This post was contributed by guest blogger Derek Simon. The actual experiments you do will be determined...implanting an electrode and non-specifically electrically stimulating an ill-defined region of the brain...trained to “freeze” upon anticipation of a mild electric foot shock that has become associated with a visual...using HR was found to block the acquisition and retrieval of contextual fear memories [5].  In a modified...other regions of the brain may be involved in retrieval of remote contextual memories. This study is an...real-time inhibition), and behavioral (remote memory retrieval) specificity. Optogenetics can be used to more...2012): 159-172.5. Goshen I, et al. Dynamics of retrieval strategies for remote memories. Cell. 2011;147...

...antibody (N206A/8 TC Supernatant). Image attribution: James Trimmer, UC Davis.   ...interact with the air-water interface (AWI) before vitrification, they can be completely denaturated (left) or...representative micrograph (C), particle orientation distribution and Fourier-shell correlation curve (D), and...protein. While the addition of LEAs may bias the distribution of particle orientations, this could be overcome...off-target editing. Cargo size limits are also less stringent for eVLPs than other viral delivery methods, allowing...anti-GFAP [N206A/8R] antibody shared by James Trimmer’s lab (Figure 5) as well as antibodies for identifying...