We narrowed to 731 results for: Sis;

...the DNA during DNA synthesis and then diluted with subsequent rounds of DNA synthesis (cell divisions)....it comes to labeling cells for flow cytometric analysis, the most common method is a cell surface label... and gate on the positive events for your flow analysis. However, certain downstream applications, like... even at room temperature, allowing for a flow analysis to be shifted to a later day. Note that intracellular... cells can still be easily gated on during the analysis. Conclusion Congratulations! You made it through...

...ddNTP to a synthesis reaction along with all four regular nucleotides results in the synthesis of DNA strands...run on a gel, and transferred to a membrane for analysis through hybridization of a radioactively or fluorescently...blotting, in conjunction with restriction digest analysis, is used to identify the presence of the mutated...sequences among DNA fragments separated by gel electrophoresis." Journal of molecular biology98.3 (1975): ...copy number changes." Protocols for Nucleic Acid Analysis by Nonradioactive Probes (2007): 205-226. PubMed...

...Cells on the membranes will then be exposed to a lysis buffer to release recombinant proteins directly ...but overnight at 30°C is a good start. Prepare a lysis solution containing your favourite bacterial protein... with thick filter paper and saturate each with lysis solution (~3 ml each), pouring off any excess. When... membranes off the plates and place each onto a lysis solution-soaked filter paper (colony-side up) and...to several freeze/thaw cycles to facilitate cell lysis: alternating between 5 min at -20°C (or -80°C) followed.... Weigele, Peter, and Elisabeth A. Raleigh. "Biosynthesis and Function of Modified Bases in Bacteria and...

...fused to the puromycin resistance gene through a 2A peptide. The puromycin resistance gene enables selection...requested plasmid, SpCas9 is still going strong as the basis for many genome editing experiments. So, without...ribosome to translate Cas9-2A and the Puromycin resistance gene as separate peptides. PX459 This is the ..., which had a point mutation in the puromycin resistance gene that made it less effective in certain cell...

... in bacteria, transposons can move antibiotic resistance genes between plasmids or from plasmids to the... how they can be used in the lab. Transposon mutagenesis screens Transposons, by their nature, are mutagenic... elements, which makes them a great tool for mutagenesis screens that detect loss-of-function or gain-...to consider when selecting a transposon for a mutagenesis screen. For example, piggyBac and Tol2 are best...Insertion of Transposable Elements by Guide RNA-Assisted Targeting) system. INTEGRATE is based on a naturally...of which has several different applications (mutagenesis screens, transgenic animals, gene transfer). ...

...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...

... One consists of NLuc fused to enhanced green fluorescent protein (GpNluc) and the other consists of NLuc...transfer (FRET) probes. These probes generally consist of a responsive protein (e.g. calcium binding protein...now specializes in supporting Promega cellular analysis products at Promega.     References 1. Hall, M.P...optical imaging and flow cytometry for monitoring tumorigenesis. Cancer Res. 75, 5023-33. PubMed PMID: 26424696... (2015) NanoBRET-A novel BRET platform for the analysis of protein-protein interactions. ACS Chem. Biol...

...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...

...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...

...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...

...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...

...yourself using PCR, but gene synthesis can save you time. Using gene synthesis, you can request the sites...before you start doing your cloning. Most sequence analysis software includes Golden Gate cloning tools that...lot of headaches down the line! Cash in on gene synthesis Before you start your cloning, it is advisable...direction (which is not easy to see in colony PCR electrophoresis), is free of undesired mutations, and that ...

...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...

...correctness of the hypothesis. The results of experiments can help support or reject a hypothesis. Looking at...Addgene or its staff. In Washington, D.C. as well as sister locations on April 22, 2017, scientists and non-scientists...knowledge. The process begins with a testable hypothesis - an educated guess about how some part of the... Magazine: A grim budget day for U.S. science: analysis and reaction to Trump’s plan Science Magazine:...

...possible by tools that allow cells to become persistently fluorescent during development. Stochastic multicolor...fluorescent neuron is visible. Chemical tags are more resistant to tissue clearing methods and can be labeled ... "Multicolor fate mapping of Langerhans cell homeostasis." Journal of Experimental Medicine 210.9 (2013...genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster." Nature methods 8.3... Snippert, Hugo J., et al. "Intestinal crypt homeostasis results from neutral competition between symmetrically...

...seven-repeat proteins: Sjuts, Hanno, et al. "Molecular basis for inhibition of AcrB multidrug efflux pump by ...system in correlation to the size of the product. Analysis of protein size and expression level can be conveniently...frequencies across timepoints or populations. This kit consists of a 48 plasmid adapter library, with each plasmid... daptomycin reveals an ordered progression to resistance." Antimicrobial agents and chemotherapy 57.11...effects during adaptive evolution of antibiotic resistance." Proceedings of the National Academy of Sciences...

...function is to regulate DNA repair, senescence, and apoptosis. Work by Kamio et al. (2025) utilized HiBiT to...characterize the proteasomal degradation of cystic fibrosis transmembrane conductance regulators (CFTR) (Kamada...ion channel, mutations of which lead to cystic fibrosis, one of the most prevalent inherited diseases.... these challenging proteins. VEGF-A mediates angiogenesis through its principal receptor tyrosine kinase...focuses on commercializing their cell and protein analysis products. You can find plasmids deposited by Promega...

...replace a gene of interest with an antibiotic resistance cassette.   Substrate DNA Design and Generation... selectable DNA fragments, such as antibiotic resistance genes, as well as non-selectable DNA fragments...red phage, λTetR, that carries the tetracycline resistance gene and the lambda red repressor cI857.  Once...confirmation of recombinant clones If a antibiotic resistance gene has been inserted, recombinants can first...first be selected via antibiotic resistance, but all clones should be further tested to confirm the presence...

...antibodies as major culprits in the reproducibility crisis of biomedical research (Baker, 2015; Bradbury &...approach. Antibody-independent methods may include analysis of transcriptomic or proteomic data from several...protein interactions could be a factor in your analysis. Example: You are planning an RNA-immunoprecipitation...and send the captured proteins off for mass spec analysis (above). When the results come back it shows that...here!  References Baker M (2015) Reproducibility crisis: Blame it on the antibodies. Nature 521:274–276...

... the cause of leaky expression. To test their hypothesis, they randomly scrambled one pair of lox sites...packaging even begins. This spontaneous recombination persists even in recombinase deficient E. coli and under...References Aschauer DF, Kreuz S, Rumpel S (2013) Analysis of Transduction Efficiency, Tropism and Axonal...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...

...guest blogger, Kaustubh Kishor Jadhav, a Research Assistant at MGMs Institute of Biosciences and Technology.... The absence of a rigid cell wall makes them resistant to antibiotics and antibacterial drugs like penicillin...of antibiotics can allow mycoplasma to develop resistance to the antibiotics. Antibiotics must only be ...prolonged use of a particular antibiotic can induce resistance for other bacteria apart from mycoplasma. Always...Jadhav! Kaustubh Kishor Jadhav, MSc is a Research Assistant at the MGMs Institute of Biosciences and Technology...

...complementary to the dsRNA (Wilson and Doudna, 2013). RNAi consists of two non-exclusive gene silencing pathways: ...different Dicer-like enzymes are involved in the biogenesis of different small RNAs (Figure 1). These siRNAs...amiRNAs based upon miRNA precursors of rice and Arabidopsis species, respectively. Short hairpin RNA (shRNA...plants, such as in the model flowering plant Arabidopsis thaliana (Li et al., 2013; Zhang et al., 2018...plants outside of limited, single cell protoplast analysis. Instead, microscopic plants must be visually ...

...extra PGC-1a1 only in their skeletal muscle show resistance to stressful circumstances – including unpredictable...Activation of the PGC-1a1 Pathway Gene expression analysis of the animals’ skeletal muscles led to another...mck-PGC-1a1 mice, and those who exercise, show resistance to depression. For the researchers led by Dr...t what they’d expected. "Our initial research hypothesis was that trained muscle would produce a substance...

... sequence similarity, phylogenetic analysis, neighborhood analysis of surrounding genes, and experimental...sequence divergence that requires a multi-pronged analysis to sort effectively. The current classification...are merely linked by a similar function. After analysis, CRISPR classifications were placed into a fairly... of factors, including your goal, experience, hypothesis, and even the time and money available. The breadth...

...FP-chimeric protein may be at any one of many stages of synthesis and degradation. For this reason it is virtually...PA-FPs by performing several rounds of random mutagenesis screening for enhanced RFP variants. PA-mCherry...negative” photoswitching. In 2008, scientists used mutagenesis screens to find a version of Dronpa that doesn't...is blocked during the Bax activation phase of apoptosis." J Cell Biol 164.4 (2004): 493-499. PubMed PMID...PMC1635685. 12.Paez-Segala, Maria G., et al. "Fixation-resistant photoactivatable fluorescent proteins for CLEM...

...higher volume can affect the lysis buffer ratio and result in incomplete lysis. You may be able to scale ..., which can happen if you leave the sample in a lysis buffer for too long or if you get a little overzealous...scale the amount of buffers used to ensure proper lysis, but you will still face column capacity limitations...troubleshooting. If you need a high level of consistency between lots, then reviewing their quality assurance...