...In this method, substrate pools were prepared consisting of one target (template) strand and four upstream...identifiable and quantifiable by capillary electrophoresis (CE) (see below). This method allowed screening...gene PCR-ligase detection reaction-capillary electrophoresis assay." Journal of clinical microbiology 45.6...mixed population of excess wild‐type DNA." Electrophoresis 34.9-10 (2013): 1415-1422. PubMed PMID: 23463388...reaction-fluorescent microsphere assay for characterization of resistance-mediating polymorphisms in African samples of...Greenough, Lucia, et al. "Adapting capillary gel electrophoresis as a sensitive, high-throughput method to accelerate...

...cytotoxicity (ADCC) c) antibody-dependent cellular phagocytosis (ADCP). Figure adapted from van Erp, et al.,... degranulation.  Antibody-dependent cellular phagocytosis (ADCP) So far, our antibodies have induced “...for is eating. In antibody-dependent cellular phagocytosis (ADCP), Fcγ receptors on macrophages bind IgG...antibodies are bound to in a process known as phagocytosis.  Other functions We’re still learning about...et al. Promoting antibody-dependent cellular phagocytosis for effective macrophage-based cancer immunotherapy.Sci...

...researchers manipulating DNA sequences on a daily basis, many found these tools inadequate for planning,... plasmids contain features such as antibiotic resistance markers and replication origins, but there is...feature. With the increasing popularity of gene synthesis, many researchers now use codon-optimized versions...control elements in commercial plasmids were inconsistent and sometimes clearly wrong. In an effort to...Additional Resources on Addgene.org Example plasmid analysis powered by SnapGene Browse All Plasmids ...

...usefulness of CPPs must be evaluated on a case-by-case basis. While setting up their CPP system to study protein...coined the term iTOP (induced transduction by osmocytosis and propanebetaine) to describe this method of...studies showed that iTOP functions through macropinocytosis. D’Astolfo et al. tested iTOP in a series...primary (hESCs) and cultured cells showed up to 70% resistance to diphtheria toxin, indicating a 70% frequency...macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles. Okada CY, Rechsteiner M. ...

...hedgehog under a new promoter each time a new hypothesis is tested. Controlling multiple genes with Gal4...Drosophila : A Fly Geneticist’s Swiss Army Knife, Genesis 34, 1–15 (2002).PubMed PMID: 12324939. 3. S. E....Davis, Gene expression systems in Drosophila: A synthesis of time and space, Trends Genet. 20, 384–391 (...Desplan, Power tools for gene expression and clonal analysis in Drosophila, 9, 47–55 (2012). PubMed PMID: 22205518...transgene expression, lineage tracing, and mosaic analysis., Cell 141, 536–48 (2010). PubMed PMID: 20434990...

...for a repair template (sister chromatid or homologue) or extensive DNA synthesis. NHEJ also finishes repair...Cheng A, Hasty P, Stark JM (2009) Limiting the Persistence of a Chromosome Break Diminishes Its Mutagenic...Ding X-Y, Guo X-P, Jin Q, Sun Y-C (2020) A CRISPR-Assisted Nonhomologous End-Joining Strategy for Efficient...Efficient Genome Editing in Mycobacterium tuberculosis. mBio 11: . https://doi.org/10.1128/mbio.02364-19 More...

...stranded and its ribose groups are susceptible to hydrolysis and heat degradation. Furthermore, RNases, or...without something to block RNase activity upon cell lysis, RNA is degraded. To effectively isolate intact ...RNases had a chance to break down RNA upon cell lysis.  In the late 1970’s, Chirgwin and colleagues showed...your samples cold until homogenization and cell lysis. This can be challenging depending on your laboratory...ways to “stabilize” RNA within tissues before cell lysis. These products, namely RNAlater®  from Thermo and...

...antibody. TRIM21, an E3 ubiquitin ligase, is the basis of TRIM-away. In normal physiology, TRIM21 recruits... to their small size (<250 nm), and studying endocytosis is further complicated by the reliance on pH-...addition, the approach allows minute-by-minute analysis of the kinetics of chromatin regulation and subsequent... reversibility allows precise and quantitative analysis of epigenetic memory at nearly any locus. The ...genes of interest. These transcription factors consist of the GAL4 or ZIF268 DNA binding domains fused...

...no small task, however, at a repository that consistently receives around 200 new DNA samples every week...inserted gene/functional mutations, antibiotic resistance, restriction sites used during cloning, and most...our other blog posts for more tips on sequence analysis, plasmid verification, and using NCBI BLAST. Tips...and read the submission instructions. As for the analysis of a large number of sequencing reactions, you... be done through the align tab of our sequence analysis web-tool. Alternatively, you can use DNA-editing...

... Your Thesis” online course.  To learn more about her site please visit www.FinishYourThesis.com.   Additional...post was contributed by Dora Farkas of www.FinishYourThesis.com and is one of many posts in our career...have trouble making decisions. In my work as a thesis coach for graduate students, I heard about supervisors...Ph.D. from MIT and is the Founder of Finish Your Thesis, an online community that has supported thousands...

...through directed evolution Hu et al. used phage-assisted evolution to generate and select for Cas9 mutants...mutants with PAM flexibility. In this system, the mutagenesis plasmid (MP6) permits quick, large-scale mutation...and subjected them to another 72 hours of phage-assisted evolution, generating xCas9 2.0-2.6. This second...where cleavage leads to the loss of spectinomycin resistance. xCas9 3.7 showed the broadest PAM range, recognizing... PAM for SpCas9. Hu et al. performed GUIDE-seq analysis to characterize genome-wide off-targets, since...

... DNA and look for potential contaminants. The analysis is a 2-step process using Geneious software: First...open-access Python scripts that can perform a deeper analysis of the data. Our serotype detection software confirms...Adeno-Associated Virus Production and Their in Vivo Persistence after Vector Delivery. Molecular Therapy 12:744...Ersing I, Haery L, Harten DeMaio K, Sanders E, Tasissa M, Kostman M, Tillgren M, Makana Hanley L, Mueller... (2020) A Novel Next-Generation Sequencing and Analysis Platform to Assess the Identity of Recombinant...

...eliminate sources of interference, but the result persisted. (Even better, multiple other labs have reproduced... AsLOV2. After several rounds of semi-random mutagenesis and screening, the EGFP and mScarlet variants...significant gains! After a few more rounds of mutagenesis, they eventually found a version of AsLOV2 with...as light-induced conformational changes are the basis of many optogenetic tools. And, since MagLOV’s parent...Magnetoluminescence from a Spatially Confined Persistent Diradical Emitter. Journal of the American Chemical...

...suppressors, and their protein products. Proto-oncogenes consist of a group of genes/proteins that, when expressed...awry. The process of programmed cell death, or apoptosis, is one such mechanism. When a cell or its genetic...project continues to evolve rapidly and, with assistance from the research community, will soon expand...pathways with potential to confer anticancer drug resistance." Science signaling 7.357 (2014): ra121. PubMed...Resources on the Addgene Blog Tackling Cancers' Drug Resistance with a New Screening Kit Learn How the Clon Tracer...

...homology, allowing the strands to anneal. DNA synthesis then fills in the gaps. The end result is a deletion...locus-specific gRNAs Step 4: Select for puromycin resistant cells Step 5: PCR amplify and sequence the locus...into the FBL locus. Upon sequencing of puromycin resistant clones, they found that 80% and 50% of clones ...References 1. Sakuma, Tatsushi, et al. (2016).  “MMEJ-assisted gene knock-in using TALENs and CRISPR-Cas9 with...and Ling Lu. (2016). “Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus...

...namely that they can cause cancer via insertional mutagenesis (which HIV does not do). Furthermore, molecular... thanks to Addgenie Rebecca Hutcheson for her assistance with lentiviral replication.  Resources and ...1974). Infectious rous sarcoma virus and reticuloendotheliosis virus DNAs. Journal of Virology, 14(5), ...polymerase of Rous sarcoma virus: Initiation of synthesis with 70 S viral RNA as template. Journal of Molecular...Full-Length Human Immunodeficiency Virus Type 1 DNA Synthesis. Journal of Virology, 82(5), 2376. https://doi.org...

...recombination.    Designing donors for HDR The basis of the HDR pathway relies on repair from a template...molecule, which is usually an endogenously available sister chromatid, though exogenous DNA can be used as ...kb), a dsDNA donor will need to be used due to synthesis limits of oligonucleotides. These donors traditionally...predominantly in S and G2 phase of the cell cycle, when a sister chromatid is likely to exist as a repair template...long. Prolonged cell cycle arrest can lead to apoptosis in as little as 10 hours, so read up on the timing...

...human CRISPRko sgRNA library The Brunello library consists of 77,441 sgRNAs with an average of 4 sgRNAs per...depletion of essential genes. In the subsampling analysis of the sgRNAs, Brunello even with only a single... a comparison study screening for vemurafenib resistance in A375 cells. The SAM system (CRISPR/Cas9 Synergistic...Mediator) uses an improved activator design that consists of inactive Cas9-VP64, a sgRNA with two MS2 RNA...reading frame (ORF) overexpression libraries for resistance to MEK inhibition. Although both Calabrese and...

...The ACE reporter is a bicistronic construct that consists of a mutated mCherry and a downstream constitutively...mScarlet reporter, luciferase reporter, and neomycin resistance reporter. Image adapted from Katti et al., 2020...As a proof of concept the Dow lab tested their hypothesis with GFP fluorescence. To do this they generated... including mScarlet-I, luciferase, antibiotic resistance markers, and other enzymes such as Cre-recombinase...proteins including fluorescent proteins, antibiotic resistance, and luciferase. References Gaudelli NM, Komor...

...available to confer greater transcriptional control, assist with proper protein folding, and deal with sub-...in outer membrane protease VII, which reduces proteolysis of the expressed recombinant proteins. lon protease...deleted (designated lon or Δlon) similary reduce proteolysis of the expressed proteins. hsdSB (rB- mB-): These...coli B strain, except ** which are K12 Strain Resistance Key Features Genotype Use BL21 (DE3)   Basic.... The pLys plasmid contains a chloramphenicol resistance cassette for positive selection and a p15A origin...