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TypeBlog Post...were developed before CRISPR-Cas9, for use in applications like siRNA and antisense oligonucleotides. Some... after less than a minute of exposure to the appropriate light. The photoactivatable counterpart to this...protocols to get your CRISPR experiment moving. Happy editing! References and Resources References Hendel...
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Hot Plasmids - March 2020 - base editors, GEVI, MoClo, and optogenetics
TypeBlog Post... changes in neural activity when paired with appropriate fluorescent optics. Existing red fluorescent ...organisms such as E. coli and yeast. Because this approach uses standardized parts, it allows for greater...increasingly been recognized for its biotechnological applications including conversion of CO2 into water and electrical... -
Celebrating 15 Years of Scientific Sharing
TypeBlog Post... work on our external website appearance and functionality are happening and this strengthens our image...support more new projects. Diversification is happening. The Addgene team will have some fun challenges... -
Tagging Optogenetics and Chemogenetics Receptors: Fluorescent Proteins and Other Options
TypeBlog Post...fluorescence that allow easy detection with the appropriate fluorescent illumination in in vivo, ex vivo,...of the most common tags currently used, are approximately 240 amino acids in size. The size of these proteins...Which tag should I use? When deciding on the appropriate tag, the experimental design should be the main... -
RaPID Detection of RNA-protein Interactions
TypeBlog Post...work. APEX2, a faster peroxidase-based labeling approach, is an alternative option but it’s toxic to tissues...subtracted from Ramanathan et al.’s RaPID datasets. Applications of RaPID RaPID sounds like a nifty technique...help answer? Ramanathan et al. outline three applications of RaPID: Discovery. To discover novel RNA-... -
dTAG - You're it!
TypeBlog Post...proteins is an exciting strategy, it is difficult to apply this technique to oncoproteins that lack selective...that are not possible with traditional genetic approaches. In addition, the dTAG system is versatile and...update with vendor and catalog numbers. As we are happy to share aliquots of dTAG-13 in the meantime, please... -
Gendered Innovations: Why Does Sex of the Cell Matter?
TypeBlog Post...and Western Europe have taken three strategic approaches to gender equality over the past several decades...Australia worked with stem cells in mice. They appropriately used male and female mice (using both sexes ... the sex of the most commonly used cell lines appearing in American Journal of Physiology-Cell Physiology... -
Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP
TypeBlog Post... Tang and Cepko were eager to see if they could apply GFP regulation to other types of proteins. They ...systems are easily adaptable to neuroscience applications, including optogenetics, and they should make...Tang note that their scaffold system is likely applicable to many types of proteins. For proteins with ... -
Antibodies 101: Stripping and Reprobing Western Blots
TypeBlog Post...protein in your sample? Or what if something weird happened during your western and your results look… funky...to probe the blot again, almost like new. This approach preserves your sample and saves you from having...retaining residual antibodies. If you have already applied antibodies and need to dry the membrane for storage... -
Plasmids 101: Modular Cloning
TypeBlog Post...multigene constructs, through multi-TU assembly,happens via three steps: Level 0: Cloning of basic parts...multi-purpose it can be. Let’s review a list of MoClo applications. Genetic circuits Most things in biology are...phenotype is best described with “it’s complicated.” Applying MoClo Interested in using MoClo? You don’t have... -
Hot Plasmids May 2018 - Optogenetics, Decaffeination, Biosensors, and Fluorescent Protein Tools
TypeBlog Post...some studies have shown that this clustering only happens under certain conditions. With its additional...protein of interest. Fusion protein oligomerization happens within seconds after a pulse of blue light, and...et. al. describe a novel system for the precise mapping of neuronal networks at the single-cell level. ... -
Split-BioID: An Improved Method for Studying Protein-Protein Interactions
TypeBlog Post...biomolecules. They can be part of multiple dynamic and overlapping complexes that have distinct functions. Many .... (2017). Split-BioID a conditional proteomics approach to monitor the composition of spatiotemporally...S., & Varnaitė, R. (2016). Meet the neighbors: Mapping local protein interactomes by proximity‐dependent... -
Adeno Associated Virus (AAV) for Cell and Gene Therapy
TypeBlog Post..., non-enveloped virus that carries a small, approximately 4.7 kb single stranded genome. AAV belongs to... over a period of 1 to 3 years – the treatment appears to be both safe and effective. Challenges for AAV...trials support the utility of AAV as a broadly applicable gene delivery tool, aspects of AAV biology should... -
A Look Back at One Year of Plasmid Sharing for COVID-19 Research
TypeBlog Post...more about it on the blog. COVID-19 research is happening fast and we’re glad to join these efforts and ... JM, DiVenere AM, Kuo H-C, Javanmardi K, Le KC, Wrapp D, Lee AG, Liu Y, Chou C-W, Byrne PO, Hjorth CK,...resources on the Addgene blog Learn more about mapping the SARS-CoV-2 - human protein-protein interaction... -
BeHeard Award 2018: Diseases of Glycosylation, Arginine Mutagenesis, & Neural Development
TypeBlog Post...Glycoimmunology group based at the Research Unit on Applied Molecular Biosciences (UCIBIO) focused on understanding...not related to this rescue function, GdnHCl was approved by the FDA in 1939 for the treatment of botulism...Given its unique molecular biology and previous approval by the FDA, GdnHCl could, in principle, provide... -
Mouse Modeling, Part 2: Breeding and Crossing Mice
TypeBlog Post...tail clip, or toe clip) and using PCR with the appropriate primers to detect your targeting vector or gene...your parental mouse should be hemizygous at the appropriate allele. When crossed to a mouse that is homozygous...F1 heterozygotes to each other will generate approximately 1 in 16 mice that harbor homozygous alleles ... -
Open Resources and Plasmid Tools For Studying C. elegans
TypeBlog Post...Scientists are currently doing this using two approaches: single-cell RNA-sequencing to identify neuronal... 2019. For more information about the aims and approach of CeNGEN, check out their publication. 3’UTRome...that can be used to simulate muscle tissue as it applies to C. elegans locomotion. To see the other resources... -
Science communication: 9 Strategies to Get Your Foot in the Door
TypeBlog Post...of competitive TED talk – to win the audience’s applause with your performance. When your institute opens...guided tours to show people where the lab magic is happening. 3) Volunteer – Step into science education Do... Blog - Hone your writing skills Take the DIY approach and start a blog. Stellar talents like Ed Yong... -
Degrading DNA with Cascade-Cas3
TypeBlog Post...Cascade binding and subsequent DNA degradation appears to be conserved. This DNA degradation portion can... portion of DNA using only a single gRNA. Cas3 appears to have fewer off-target alterations, likely due...Mashimo, T. (2022). Genome editing technology and applications with the type I CRISPR system. Gene and Genome... -
PRIDICT: Predicting Efficiencies of Prime Editing Guide RNAs
TypeBlog Post...machine learning (PRIDICT, PRIDICT2.0). How to apply PRIDICT in prime editing experiments Prediction ...pegRNA efficiencies with pridict.it. This approach leverages the strengths of PRIDICT/PRIDICT2.0 ... computational biology. His expertise includes applying machine learning techniques to make sense of biology... -
Truncated gRNAs for Regulating Gene Expression
TypeBlog Post...virtually any genetic locus when complexed with an appropriately designed small RNA, or guide RNA (gRNA). The...expression with truncated gRNAs An alternative approach is to modulate Cas9’s nuclease activity by modifying...engineering parameters via a library-on-library approach." Nature methods 12.9 (2015): 823-826. PubMed ... -
3D Printing Meets CRISPR Cas9
TypeBlog Post...of proteins. But they are still flat. To truly appreciate the 3D structure of these proteins, it is necessary...molecular viewing software. In reality, this rarely happens. But if you ever have an opportunity to read one...invariably say something like “Oh,…I didn’t quite appreciate that….” For example, when Tom Steitz was presented... -
Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR
TypeBlog Post... original CAPTURE and CAPTURE 2.0 methods. Applications of CAPTURE 2.0 CAPTURE 2.0’s increased rate ... of chromatin interaction networks A second application for CAPTURE 2.0 is the construction of spatial...temporal changes in chromatin interactions A third application for CAPTURE 2.0 is detection of temporal changes... -
Tips for a 1st time AAV user (by a Rookie AAV user)
TypeBlog Post...Addgene AAV Production protocol, I take a lazier approach for harvesting my AAV. I skip the PEG precipitation...the PEG will separate into two phases. If this happens, the solution can be cooled and the two phases ...immediately before iodixanol purification. This approach gives me a bit of flexibility since I can store... -
Hot Plasmids and Viral Preps - May 2021
TypeBlog Post...The CRISPR corner New ready-to-use viral preps Mapping antibody escape in SARS-CoV-2 by mutational scanning... sequencing. From Starr et al Cell., 2020. (B) Mapping antibody-escape mutations with the yeast display...specific anti-SARS-CoV-2 antibodies (Fig 1B). This approach can augment viral surveillance by enabling rapid... -
Fluorescent Proteins 101: Visualizing Subcellular Structures & Organelles
TypeBlog Post... map the internal organization of a cell. This mapping can be done by using antibodies targeted to the...meaningful way it is important to choose the appropriate quantification methods and tools. It is usually...Pages 2. Cooper GM. 2000. The Cell: A Molecular Approach. 2nd Edition. 3. CureFFI.org. Cell Biology 04... -
Plasmids 101: Optimizing Plasmid Yields
TypeBlog Post...are, you’ll even have DNA left over for other applications, like PCR, cloning, transfection, or long-term...wash step or use a different strain. For most applications, Addgene uses and recommends DH5𝝰! This strain...your E. coli to ensure it is suitable for your application. We have previously reviewed many common lab ... -
PITChing MMEJ as an Alternative Route for Gene Editing
TypeBlog Post...(2015). “A Scalable Genome-Editing Based Approach for Mapping Multiprotein Complexes in Human Cells.” ...lies in the diversity of nucleases and their applications. It will be interesting to see what new editing... -
Tips for Using BLAST to Verify Plasmids
TypeBlog Post...reference sequence into the Subject Sequence box that appears. Aligning two nucleotide sequences is probably ...frameshift mutation present in the ORF is readily apparent when viewing blastx results. When using blastx...webservices/#pairwise_sequence_alignment_psa Happy BLASTing! Do you have any tips for using BLAST to... -
Molecular Biology Protocol - Restriction Digest of Plasmid DNA
TypeProtocol...of interest (see below for recommend amounts) Appropriate restriction enzyme (see manufacturer's instructions...instructions for proper ammount) Approrpriate restriction digest buffer (see manufacturer's instructions) ...as Addgene's Sequence Analyzer . Determine an appropriate reaction buffer by reading the instructions for...The amount of DNA that you cut depends on your application. A diagnostic digest typically involves ∼500 ...volume usually varies from 10-50 µL depending on application and is largely determined by the volume of DNA...better. Mix gently by pipetting. Incubate tube at appropriate temperature (usually 37 °C) for 1 hour. Always...manufacturer’s instructions. Pro-Tip Depending on the application and the amount of DNA in the reaction, incubation...