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TypeBlog Post...constructs with AAV. In the case of channelrhodopsins, fewer channel proteins are required for the transduced...these tubes. Rinse off the filter - When using a concentration filter column, make sure to rinse off the surface...our Concentrating AAV Protocol (Step 10) for instructions on how to do this. Concentrating your virus ...necessary to visualize expression of fluorescent proteins, for example, as many viral particles must infect...cell to become light responsive than fluorescent proteins required to get a good fluorescent signal. It ...
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Addgene Welcomes John Doench to the Advisory Board
TypeBlog Post...the Broad Institute here Read other blog posts about CRISPR Learn more about the Broad Institute and the...the Genetic Perturbation Platform at the Broad Institute and has worked with many Addgenies to help improve...research is still relevant to his work at the Broad Institute today, where he helps researchers in a variety...own research questions. Starting at the Broad Institute For his post-doc, Doench worked on using RNAi... School and then applied to work at the Broad Institute: “The model that the Broad was building that had...development under the Platform model at the Broad Institute because they are “not just developing technology... -
A Deep Dive into BioBuilder
TypeBlog Post...students in real-world lab activities? I’m trying to inspire them and also keep up with all the new biotechnology...being asked in MIT’s research labs, BioBuilder inspires students and teachers to apply engineering principles...final project. BioBuilder’s open-access curriculum begins with students reading comic strips and watching...droppers, centrifuge tubes, circuit parts, and lab-strains of bacteria to study — to conduct their hands-on...breakdown, production of fire-resistant housing insulation, and cleaning up salt runoff from winter icing.BioBuilderClub...summer teacher professional development program that trains nearly 100 teachers each year, with workshops held...BioBuilder's open-access curriculum available on Amazon Inspire. Many thanks to our guest blogger, Mary Tamer... -
Quick Guide to Working with Drosophila Part 3: Genome Engineering in Flies
TypeBlog Post...geneticists to study gene function. In this final installment, I’ll provide a brief introduction as to how ...overexpression and knockdown. Addgene also has a number of insect vectors to consider. An easy way to see what is...available is to browse Addgene’s Collection under the insect expression system or by filtering by fly as species...which locus your transgene ends up (1): Random insertion has the advantage that you can get varying expression... inherited on the same chromosome. Targeted Insertion allows for direct comparisons between lines expressing...would you like your flies ready for use? Random insertions take longer because you need to determine in ...chromosome lines but is not necessary with targeted insertions (here you would know beforehand on which chromosome... -
Plasmids 101: Transformation, Transduction, Bacterial Conjugation, and Transfection
TypeBlog Post...take a look at these different methods of DNA insertion. Transformation Transformation is the uptake ...inject their genetic material into the cell. Once inside, phages can follow one of two different life cycles...particular integration site. The integrated phage remains dormant until it is triggered to enter the lytic...referred to as transduction. Transferred DNA once inside the infected bacterium can either exist as transient...commonly use phagemids, a DNA cloning vector that contains both bacteriophage and plasmid properties. The...recombination between two nutritional deficient E. coli strains that resulted in a wild type E. coli (Griffiths...bacterium through direct contact. The donor bacterium contains a DNA sequence called the Fertility factor (F-... -
Viral Vectors 101: Gamma-Retroviral Packaging Systems
TypeBlog Post...packaging (Gag, Pol, and Env), the isoforms of these proteins, as well as the viral LTRs, differ. As a result...developed by Garry Nolan at Stanford (Swift, 2001) contains Gag-Pol and either an ecotropic envelope, Phoenix-ECO... just a few days. Another variant, Phoenix-gp, contains only Gag-Pol and enables additional flexibility...transduction are not encoded by the transfer plasmid, but instead are provided in trans by other plasmids or the ...Cloning capacity between the LTRs is ∼8.5 kb, but inserts bigger than ∼3 kb are packaged less efficiently...found at both the 5' and 3' ends of the provirus). Contains sequences necessary for activation of viral genomic... Acts as an RNA pol II promoter. The transcript begins, by definition, at the beginning of R, is capped... -
Viral Vectors 101: Viral Applications
TypeBlog Post...variety of cargo, and even handle a variety of terrains. They are, in fact, a choice delivery vector from...permanently. Viral vectors can also be used to express proteins. Viral expression cassettes typically contain ...from scratch! Why deliver these systems virally instead of by another method? Viral delivery is attractive...thousands of elements simultaneously in your system instead of just one at a time. Many of these libraries ... are packaged and delivered with viral vectors instead of a traditional transfection method. This is because...the actual virus to which immunity is desired. Instead, components of the virus are now delivered in other...immunogenicity of attenuated, nef-deleted HIV-1 strains in vivo. Retrovirology. 2007 Sep 23;4:66. doi: ... -
Mycoplasma Contamination: Where Does It Come From and How to Prevent It
TypeBlog Post...Kaustubh Kishor Jadhav, a Research Assistant at MGMs Institute of Biosciences and Technology. If you are reading...subculturing processes or from the air if the cell line remains exposed for a longer time. The aerosols are invisible... be detected by a luminometer. Non-specific DNA stains can be added in the infected culture medium to ... DNA staining alternative. This method uses DNA stains like DAPI and Hoechst 33258 to stain all DNA. Some...sneezing or coughing in the lab. The human mouth contains a wide variety of mycoplasmas and these can enter... sure to change the gloves and masks regularly instead of keeping them in the pockets of your lab coats...you keep all the required equipment and reagents inside the laminar airflow before and while you are working... -
RANbodies: Reporter Nanobody Fusions
TypeBlog Post...produced in animals instead of via recombinant DNA, they can’t be genetically tagged and instead are conjugated...reporter consists of two chicken IgY domains. Chicken IgY domains are especially useful for multi-color...’t require a secondary antibody for detection. Instead they are detected with tyramide signal amplification...antibody and a ≥100-fold increase in signal over intrinsic fluorescence. Increased signal is likely due to... et al can be found here. Can’t find a RANbody against your favorite protein? No worries! As more nanobodies... -
All in a Twist: dsRNA
TypeBlog Post...target pathogenic proteins, as well as in the lab to deplete expression of proteins of interest. RNAi ...found (typically RNA that the RNAi is designed against), RISC can repress translation and/or degrade target...agriculture as species-specific pesticides and insecticides, an advancement over previous chemical versions... these structures can in turn lead to genome instability. Thus, it’s important to strike a balance between..., I. X., Grunseich, C., Fox, J., et al., Human proteins that interact with RNA/DNA hybrids. Genome Res...10.1101/gr.237362.118 Chen, G. Y., Hur, S. Cellular origins of dsRNA, their recognition and consequences. Nat...