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TypeBlog PostPublishedJune 29, 2021, 1:15 p.m....hybridoma, you can choose one that produces high-affinity/high-avidity antibodies. The same hybridoma is...drive can be identified by sequencing the heavy and light chains. To circumvent any possible genetic drift... Figure 2: Changes to the heavy chain or light chain genes within a hybridoma impacts the specificity...validating monoclonal antibodies, their cost is higher than that of polyclonal antibodies. For example...
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The Many Reasons for Irreproducible Research- and a Vaccine to Eradicate It
TypeBlog PostPublishedJan. 30, 2020, 2:15 p.m....cognitive biases such as motivated reasoning and hindsight bias, we may fool ourselves and our readers into...replications are unavoidable, but can be minimized with high powered research designs. Unanticipated discoveries...become public eventually). It is typically done right before data collection starts, and should include...example, if you hypothesize that growth rates will be higher in treatment versus control, and you think there...of the study, say a fifth time has unpredictably high rates, should be reported as a new discovery that... -
Reproducibility for Everyone: Lessons from an Open Science Collaboration
TypeBlog PostPublishedFeb. 28, 2019, 1:34 p.m.... and international team assembled the right people at the right time. Be inclusive and seek solutions...Teytelman’s vast network and ability to identify the right opportunities, he was able to bring together talented... email the team. Acknowledgements One of the highlights of 2018 for me was to collaborate with this incredibly... -
Hot Plasmids April 2018 - Protein Degradation, Nanoscopy, FIRE-Cas9, and Yeast Expression Tools
TypeBlog PostPublishedApril 23, 2018, 2 p.m....Every few months we highlight a subset of the new plasmids in the repository through our hot plasmids...Chemistry for his contributions to super-resolution light microscopy, comes from his recent Nature Communications...difficult to study in living cells using conventional light microscopy due to their small size (<250 nm), and...probes had to target endosomes, be exceptionally bright (which is necessary for effective STED), and be... -
Plasmid Tools for Microbiome Studies
TypeBlog PostPublishedJune 27, 2019, 12:25 p.m....growth of diverse species. These results further highlight the need for in situ genetic tools for microbiome...Yeast Toolkit by replacing the backbone vector’s high-copy number bacterial ColE1 origin with the RSF1010...microbial population dynamics in the gut can provide insight into how the microbiome responds to infection, ...on the assembly of fluorescent proteins into one bright particle that segregates into one cell during cell... -
Supporting Rare Disease Research with the 2020 BeHEARD Awards
TypeBlog PostPublishedSept. 8, 2020, 2 p.m....research. Congratulations to the winners! Below, we highlight their research and how they will use plasmids ...research into CCDS. The brain and muscles have a high metabolic demand that is powered by ATP. A molecule...bicuspid aortic valve at a rate that is 50-100 times higher than women in the general population,” says Dr.... and can lead to cardiac developmental defects. Eight genes from the X chromosome are expressed at significantly... -
Giving gRNAs a Facelift - Synthetic and Beyond
TypeBlog PostPublishedOct. 20, 2022, 1:15 p.m....the entire editing system with a simple LED light. Lighting up your gRNA with fluorescent dyes In a scientific... nucleic acids with N-methyl substitutions are slightly more efficient and less toxic to mammalian cells...less than a minute of exposure to the appropriate light. The photoactivatable counterpart to this system...unable to engage a target sequence. Upon a brief light exposure (several seconds) the gRNA is released ... -
To Each HIS Own
TypeBlog PostPublishedMarch 2, 2023, 2:15 p.m..... The protein of interest is then eluted using a high imidazole buffer that outcompetes the interaction...protein but not that of the protein of interest. A high imidazole elution buffer disrupts the interaction...concentration of imidazole used in the wash buffer should be high enough to disrupt contaminant-metal ion binding ...therefore more selective. When using systems with higher levels of histidine-containing protein contamination... histidine residues that are present, the more tightly the protein will bind to the metal ion substrate... -
Antibodies 101: Secondary Antibodies
TypeBlog PostPublishedDec. 21, 2021, 2:15 p.m....direct approach (left) versus the indirect approach (right). Primary antibodies are represented in blue; secondary... purple; signaling molecules are represented in light green; and proteins are represented in orange. Created...only benefit of secondary antibodies. Finding the right primary antibody for your assay and protein of interest...antibodies are different species and/or isotypes. For highly multiplexed assays, using secondary antibodies ... gel-based method identifies proteins solely by weight and size, relying on the signaling molecule solely... -
Transferable Skills Guide: Cross-team Communication
TypeBlog PostPublishedNov. 21, 2019, 1:53 p.m....conference you’re attending and who approaches you. You might also have cross-team projects within your own lab... errors in our LIMS tracking system. Though you might not have to communicate with a team of software ...document, I update the training frequently. In hindsight, getting a list of frequently used words like ...everyone on the same page. Others in the meeting might also have the same question and asking can help ..., meetings will be shorter and more productive. Higher Productivity. Taking active measures in improving... -
Pairing CombiGEM and CRISPR for Combinatorial Genetic Screening
TypeBlog PostPublishedApril 12, 2016, 2:30 p.m....Methods to scale up multiplexed CRISPR systems for high-throughput screening are vastly useful for mapping...CombiGEM uses iterative one-pot reactions with straightforward restriction digestion and ligation steps to... by sequencing its set of barcodes. CombiGEM is highly flexible and can accommodate any genetic elements...Central PMCID: PMC4253859. 4. Hart, Traver, et al. "High-Resolution CRISPR Screens Reveal Fitness Genes and...PMC3972032. 10. Wong, Alan SL, et al. "Massively parallel high-order combinatorial genetics in human cells." Nature... -
Plasmids 101: Golden Gate Cloning
TypeBlog PostPublishedAug. 27, 2015, 2:30 p.m...., these problems can be overcome by screening a higher number of potential clones. Golden Gate assembly...constructs with an attB recombination scar encoding eight amino acids, but Golden Gate assembly can be designed...that may not be amenable to "domestication", you might want to consider using an alternative method like... for cloning complicated constructs in a single, high-efficiency step. References: Sequential amplification...one pot, one step, precision cloning method with high throughput capability. Engler C, Kandzia R, Marillonnet... -
Antibody Validation for Flow Cytometry
TypeBlog PostPublishedSept. 10, 2024, 1:15 p.m....cell lines to the -omics data. The cell lines with higher expression of the target protein, as determined...expression, are often preferred for this approach. A highly selective antibody should be able to detect even...only be able to detect the target protein at the high levels caused by overexpression. Additionally, if...clones The antibody validation process represents a high level of effort and time, which may not be available... & ABIF Consortium. (2024). A guide to selecting high-performing antibodies for Synaptotagmin-1 (Uniprot....15467 Virk, H. S., Rekas, M. Z., Biddle, M. S., Wright, A. K. A., Sousa, J., Weston, C. A., Chachi, L.... -
Rabies and Neuronal Tracing
TypeBlog PostPublishedMay 29, 2018, 1:51 p.m....these high levels of viral replication are toxic. Second, infecting subsequently higher and higher order...transmission occur, spreading the virus through higher and higher order neural connections. While this transsynaptic... -
Lab Automation at Addgene
TypeBlog PostPublishedMarch 22, 2018, 1:28 p.m....Updated Apr 6, 2021. If you’re doing high throughput work, it’s likely that you come into the lab wishing...transformation One protocol that has been automated is high throughput transformation. With our ever growing...the deck of the Biomek. The FXP id also able to do high throughput DNA preps and other molecular work if... through this process. With enough agar and the right program, the liquid handler can fill 400 vials in...hand. How can you start automating your lab? You might not be doing enough transformations, minipreps, ... -
Quickest Way to Deposit Plasmids: The Deposit Spreadsheet
TypeBlog PostPublishedMarch 8, 2017, 3:15 p.m....above, or other. High or Low Copy Choose from: high or low copy. Choose “high copy” if a sufficient...standard cloning strain DH5α. For plasmids with highly repetitive sequences (prone to recombination in...Example of the deposit spreadsheet showing the first eight columns and a pop-up instruction box for the “Purpose... -
Open Resources and Plasmid Tools For Studying C. elegans
TypeBlog PostPublishedJuly 18, 2019, 12:55 p.m....expression and degradation. These workshops not only highlighted new tools but also included time for questions...presented at the C. elegans meeting. Here I’ll highlight a few toolkits that we have available at Addgene..., and Erik M. Jorgensen. "SapTrap, a toolkit for high-throughput CRISPR/Cas9 gene modification in Caenorhabditis... -
Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene
TypeBlog PostPublishedApril 6, 2021, 12:30 p.m....possible by the use of our high-throughput DNA isolation process that yields high-quality isolated DNA samples... on our ability to provide requesting scientists high quality data! Addgene's new in-house NGS process...be familiar with our efforts to ensure access to high quality, ready-to-use viral preps and pooled libraries...Konermann S, Gootenberg JS, Abudayyeh OO, Platt RJ, Brigham MD, Sanjana NE, Zhang F (2017) Genome-scale CRISPR-Cas9... -
A New Generation of Adenine Base Editors Improves Editing in Primary Human Cells
TypeBlog PostPublishedMay 7, 2020, 1:15 p.m....of the ABE8s ranged from 1.5x higher for positions A5-A7 to 3.2x higher for positions A3-A4 and A8-A10...synthetic library of TadA sequences to create the eighth generation of adenine base editors, called ABE8... in variants named “ABE8.x”: “8” stands for the eighth round of ABE evolution and the “x” is the placeholder...editing frequencies. For applications that require high DNA editing specificity, for example for potential... -
Early Career Researcher Toolbox: Finding Relevant Papers
TypeBlog PostPublishedJan. 9, 2020, 1:45 p.m....installment of the Early Career Researcher Toolbox will highlight four tools for finding journal articles related...summary of the topic and a chronological list of highly cited papers that contain your search term. When...of the page, as well as the number of citations highly influenced by this paper. Influence is measured...number of publications that heavily cite, or are highly influenced by the author. You can also see who ...