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TypeBlog Post...TRIM21 recruits ubiquitin and the proteasome to destroy antibody-bound pathogens. Clift et al. found that...necessary for studying endosomes, the Hell lab designed their super-resolution pH indicators (SRpHi) biosensors...sgRNAs are used with FIRE-Cas9, experiments can be designed to target multiple loci at once. In addition, ...
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Human Germline Editing Using CRISPR
TypeBlog Post...donated skin, blood and semen samples. Ma et al. designed plasmids containing Cas9 and two different gRNAs... at 3 days post fertilization. Since Ma et al. designed a repair template with two synonymous mutations...thus quieting some fears about the creation of “designer babies.” The differences in editing between iPS... -
CRISPRainbow and Genome Visualization
TypeBlog Post...ortholog requires a different PAM sequence, a gRNA designed for one dCas9 should be specific to that ortholog...boxB Magenta boxB-MS2 White boxB-MS2-PP7 Despite the success of Ma et al.’s approach, fluorescently...genome editing. Such a system has been previously described for transcriptional activation/repression and ... -
Antibodies 101: Polyclonal Antibodies
TypeBlog Post...is tested following each immunization until the desired level of antibodies is reached - typically within...within 2-4 months. After the animal has the desired level of antibodies in its bloodstream, the antibodies...species. Pre-adsorbed polyclonal antibodies are designed for experiments where antibodies raised in several... -
Viral Vectors 101: An Introduction to AAV
TypeBlog Post...serotypes The names of AAVs contain two numerical designations, separated by a slash, that indicate the ITR...interested in expressing multiple small genes, you can design single rAAV vectors with multiple genes packaged... an option (McCarty et al., 2001). This AAV is designed so that the single stranded DNA folds back on ... -
Transferable Skills Guide: Public Speaking
TypeBlog Post...you labeled your gene of interest incorrectly on slides 8-16. Your notes disappear. The wrong version of... speaking is hard. Aside from the diving board charades, I doubt anyone but me remembers any of the incidents... biomedical field, with very little text on the slides and notes instead of written speeches. When you... -
Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP
TypeBlog Post...GFP-labeled cells. Once Tang and Cepko found a description of GFP-binding nanobodies, the project really...stable inside cells. Nanobodies binding GFP were designed in 2009. Subsequent work showed that nanobodies...green-red Cre-lox systems. T-DDOGs can also be designed with other DNA-binding domains, including the ... -
The Future of Research Symposium Boston 2015
TypeBlog Post...growing dialogue on the state of a system that, despite all of its successes, has many deep-rooted flaws...all bad, however - there are some signs that the tides may be turning. In 2013, the NIH announced its first...in Scientific Training (BEST) awards, which are designed to support new approaches that address the need... -
Viral Vectors 101: Calcium Sensors
TypeBlog Post... There are several calcium reporters specially designed for the aims and requirements of in vivo studies...populations of interest. Many of the calcium sensors described are also already packaged into viral vectors and... L, Campbell RE. Structure-and mechanism-guided design of single fluorescent protein-based biosensors.... -
CRISPR 101: Targeting Non-Coding RNAs with CRISPR/Cas9
TypeBlog Post...non-coding RNAs (lncRNA). lncRNAs are over 200 nucleotides in length and lack an open reading frame, so ...transcription machinery of a target gene or DNA that codes for ncRNA. CRISPRa, on the other hand, recruits ...which targets RNA instead of DNA and selectively degrades RNA molecules. This system is often used to knock... -
Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene
TypeBlog Post...throughout the library preparation process and includes Picogreen quantification of various intermediate...sequences for Illumina sequencing as well as unique barcodes per sample so we can assign sequencing reads to... use a modified version of the Python program described by Joung et al. 2017, which we hope to make available... -
Tips for Technical Support Calls
TypeBlog Post...plasmids and pooled libraries were originally described in a peer-reviewed scientific publication. We ...scientists answering their technical support questions (besides Addgene, I know both Abcam and NEB have Ph.D.s ...believe that scientists who contact customer service deserve prompt, courteous responses. No one is likely to... -
5 Tips for Troubleshooting Viral Transductions
TypeBlog Post... try amplifying the viral vector with bacteria designed to minimize rearrangements such as NEB Stable ...production of virus. Alternatively, if the virus encodes a fluorescent protein, the packaging cells could...working with, there’s a lot that goes into virus design, production, and transduction. Once you’ve harvested... -
Starter guide to induced pluripotent stem cells (iPSCs) part 1: A renaissance in regenerative medicine
TypeBlog Post...essence, pluripotent cells can be thought of as descendants of totipotent cells. An example of pluripotent... produced Ethical concern High. Embryos are destroyed during ESC isolation. Low Chances of post-transplantation...R., M.W. Beckmann, and W. Wurfel, Non-embryo-destructive Extraction of Pluripotent Embryonic Stem Cells... -
Antibodies 101: Conventional vs Spectral Flow Cytometry
TypeBlog Post...positive and negative populations, Careful panel design In both conventional and spectral flow cytometry...indistinguishable. This tool is of particular value when designing a panel. Figure 3: Similarity index...if markers are co-expressed in your cells when designing your panel. If you have two highly overlapping... -
The AAVantages of AAV in CRISPR Screens
TypeBlog Post... This vastly increases the possibilities when designing screens. Each serotype has its own tropism, or...experiment. Additional considerations One of the downsides to using AAV is the limited packaging capacity...Adames, A. C., Saur, D., & Rad, R. (2022). Tutorial: design and execution of CRISPR in vivo screens. Nature... -
Transgenic Organisms, Cas9 Gene Drives, and Appropriate Safeguards
TypeBlog Post... also the easiest: don't use a DNA vector that encodes both Cas9 and sgRNA. What if I want to work with...one mistake can be enough. Reversal drives are designed to overwrite a previous gene drive and thereby... on popular support, which in turn depends on widespread trust in scientists to develop new technologies... -
Tips for Using BLAST to Verify Plasmids
TypeBlog Post...terrific getting-started guide for BLAST, which includes a simple explanation of the different BLAST programs...matrix BLOSUM62, but you may want to check the description of the other matrices to see if another would...alternatives Depending on your sequencing result and desired analysis, BLAST may not always be your optimal ... -
SciComm: Taking Science to Elementary and Middle Schools
TypeBlog Post... can teach 5-10 year olds in a single evening. Grades K-5 cover a broad range of attention spans, skill...CRISPR work? This section was essentially background slides getting everyone on the same page in terms of the... by running it by the teachers and sharing some slides on Twitter, where I was able to get some great ... -
Plasmids 101: Secondary Nanobody Toolbox
TypeBlog Post...their production requires animal sacrifice and precludes genetic engineering of a tag or fusion with a ... with secondary nanobodies conjugated with the desired fluorophores. In figure 4B below, triple colocalization...review. If you just want to use the nanobodies described in Pleiner et al, the key steps for generating...