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We narrowed to 608 results for: Sis

Showing: 41 - 80 of 608 results

  1. The Many Reasons for Irreproducible Research- and a Vaccine to Eradicate It

    Type
    Blog Post
    Published
    Jan. 30, 2020, 2:15 p.m.
    ...the forefront in the so-called “reproducibility crisis.” Results in preclinical, biomedical and psychological...be able to look under the hood at the raw data, analysis code, and other materials. The use of reporting...collected, what the hypotheses are, and how each hypothesis will be tested with a specific statistical test...Preregistration makes clear the distinction between hypothesis testing work, in which a-priori hypotheses are...are tested with a new experiment; and hypothesis generation, in which data are investigated to look for...unexpected trends or differences between groups. Hypothesis generation is critical for advancement, but we...too often encouraged to present that work using hypothesis testing statistical tools that generate a p-value...

  2. 27 Hot Plasmids from 2016

    Type
    Blog Post
    Published
    Dec. 22, 2016, 3:03 p.m.
    ...including but not limited to cell growth and drug resistance. Recently, the Wu Lab at Texas Tech developed...Virus (WNV) induced cell death. The Wu lab library consists of 77,406 individual gRNAs targeting a total of...approved drugs against the entire kit; a parallel analysis that successfully identified new, highly specific...alone involves much literature searching, DNA synthesis, and plasmid assembly. Luckily, researchers from...Moduclar Cloning (CIDAR MoClo) Parts Kit. This Kit consists of a variety of plasmids containing many different...processes such as differentiation, proliferation, and apoptosis. In humans, mutations that permanently activate...plasmids to the scientific community. This collection consists of plasmids containing genes from a crowd-sourced...

  3. Cancer and the Immune System: Deciphering the Relationship

    Type
    Blog Post
    Published
    March 14, 2017, 2:30 p.m.
    ...mechanisms of the host. The cause, manifestation, diagnosis, recurrence, and treatment of cancer have been... process analogous to the development of drug resistance in bacteria, the tumor cells that escape the ...is a region of incessant chemical activity, and consists of other cell types, especially immune cells and... promotes the production of VEGF to increase angiogenesis around the tumor. This in turn regulates the... tumor cells spreading to other organs during metastasis. Evading Recognition Tumor cells over-express...damage healthy cells, and often cannot eradicate metastasis or eliminate the probability of a relapse. Chemical...the immune cells that aid tumor formation and metastasis, block the cell-surface proteins that help escape...

  4. Mesothelioma - Causes, Symptoms, and Treatment

    Type
    Blog Post
    Published
    Dec. 13, 2016, 3:30 p.m.
    ...time from first asbestos exposure to the time of diagnosis) is about 35 years, but it usually ranges from...Stage 4 cancer. These later stages of a cancer diagnosis indicate, for Stage 3, that the cancer is large...originated to another organ in the body. Such misdiagnosis and late discovery mean that most mesothelioma...mesothelioma patients have an extremely poor prognosis. Together, the cause, long latency period, and rarity of...options: Early detection, immunotherapy, and angiogenesis inhibitors New research is also going into two...the best ways to improve the traditionally dire prognosis for mesothelioma patients is to catch the disease...mesothelioma much earlier, thus improving the patient’s prognosis significantly, but also to monitor those who may...

  5. Antibodies 101: Introduction to Gating in Flow Cytometry

    Type
    Blog Post
    Published
    April 4, 2024, 1:15 p.m.
    ...for the analysis. (C) A more liberal gate allows more cells to be considered for the analysis. Depending...duplicate events, and the Live/Dead gate to focus your analysis on what’s biologically active. After that, the... of tissue processing prior to running my flow analysis, I can however clearly identify my lymphocyte ...good to ask around for advice before running your analysis. In this case, I and other lab members had worked...acquisition voltage (not shown) used in my flow analysis I can spot my lymphocyte population distinct from...beginning should not have a great impact on your analysis, and I often choose a looser gate here. I am not...worried about including irrelevant events in my analysis, as these will get excluded through further, more...

  6. Plasmids 101: Mammalian Vectors

    Type
    Blog Post
    Published
    March 25, 2014, 3:15 p.m.
    ...plasmid contains a cassette that will make cells resistant to a toxin), whereas negative selection would ...markers are listed below: Name Gene Conferring Resistance Cell Types* Mode of Action** Working Concentation...CHO, 293HEK, Jurkat T cells Blocks polypeptide synthesis at 80S; inhibits chain elongation 100-800 ug/mL...NIH3T3, CHO, Jurkat T cells Blocks polypeptide synthesis at 80S; inhibits chain elongation. 50-500 ug/mL...HeLa, 293HEK, Jurkat T cells Inhibits protein synthesis; premature chain termination 1-10 ug/mL Zeocin...reagent). Cells should die within 3-5 days and resistant colonies appear in about 10-14 days depending ...confusing since the neo/kan gene confers G418 resistance; however, like gentamicin, neomycin is typically...

  7. Scientific Reproducibility - Focusing on Solutions at the Minisymposium on Reproducibility

    Type
    Blog Post
    Published
    May 18, 2018, 7:32 p.m.
    ...data for a hypothesis and presents only the results that align with the original hypothesis. Other practices...steer clear of saying we have a reproducibility “crisis” on our hands and instead provide a brief conceptual...in-depth information about reagents, protocols, analysis tools, and more. Being that this is the Addgene...larger and larger datasets and use computer based analysis techniques, an important component of reproducibility...researchers to share code they’ve used for data analysis while other organizations like Dryad make it easier... stories with links of causation from initial hypothesis through validation are easier to understand. ...publication record will then show that the tested hypothesis is correct (a great success story) even though...

  8. Gendered Innovations: Why Does Sex of the Cell Matter?

    Type
    Blog Post
    Published
    July 21, 2016, 2:30 p.m.
    ...integrating sex and gender analysis into research. Why are sex and gender analysis important? Doing research...and gender analysis and 2) provides case studies with concrete examples of how gender analysis leads to ...harnesses the creative power of sex and gender analysis for innovation and discovery. Tackling sex and... the potential to save lives and money. A 2014 analysis of the US Women’s Health Initiative Hormone Therapy.... While most of the results were positive, the analysis did find 263,000 more osteoporotic fractures. ... learn sophisticated methods of sex and gender analysis. Universities need to incorporate these methods...harnessing the creative power of sex and gender analysis for innovation and discovery. She is a member ...

  9. Corralling and Cornering the Goldilocks Antibody

    Type
    Blog Post
    Published
    Aug. 9, 2022, 1:15 p.m.
    ...antibodies are, they are in a midst of a crisis. The “antibody crisis” came to light as antibodies became ...2015 review analyzing and explaining the antibody crisis states that up to 50% of all commercially available...Plückthun, 2015). To avoid being caught in the antibody crisis, one must corral and corner the goldilocks antibodies...scientists to confidently choose reliable and consistent antibodies for specific markers. The Screening...determine the antibody’s reliability through their own analysis of the background noise, distinct imaged pattern...bring us one step closer to resolving the antibody crisis. The mAb3D Atlas is a public service to the science...please contact us here! Ava Shipman is a research assistant in lab of Zhuhao Wu at the Icahn School of Medicine...

  10. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    Published
    Dec. 23, 2015, 3:30 p.m.
    ...functional antibiotic resistance. Colonies with an insert in pUCXKT will therefore have resistance to both kanamycin... the lambda phage integrase system which is the basis for Gateway cloning. The combination of all 11 att...activator protein to drive transcription. The anchor consists of dCas9 fused to CIB1 and is targeted to specific...readily modified to use different antibiotic resistances, restriction enzymes and plasmid backbones. pUCXKT...nonfunctional truncated version of the kanamycin resistance gene (missing the first two codons). Two stop... promoter to prevent leaky expression of this resistance. Unlike previous systems, pUCXKT does not result...containing the missing codons from the antibiotic resistance cassette, a ribosome binding site, SacI restriction...

  11. Technical Design of a Western Blot

    Type
    Blog Post
    Published
    Aug. 22, 2024, 1:15 p.m.
    ...before your enzyme lysis. For cell cultures, enzyme lysis is typically sufficient.  Lysis buffer When choosing...specificity, lower cross-reactivity, and are more consistent lot-to-lot. The highest reproducibility comes...choosing your lysis buffer, consider the subcellular location of your protein. Proteins located in membrane-bound...mitochondria or nucleus, will likely require a harsher lysis buffer than proteins easily available in the cytoplasm...or whole-cell lysate. A harsher, SDS-containing lysis buffer, like RIPA, should be considered if your ...membrane-bound protein. There are many different lysis buffers available commercially, and most common ...use of hazardous chemicals, and are not always consistent. Precast gels, while expensive, reduce protocol...

  12. Harnessing Bacterial Toxins for Allelic Exchange

    Type
    Blog Post
    Published
    Aug. 15, 2019, 12:30 p.m.
    ...chloramphenicol-resistant strains, we have also included versions with gentamicin-resistance (amplified from...disrupt expression of a gene with an antibiotic resistance cassette. This leaves a “scar” in the chromosome...be accomplished by selecting for an antibiotic resistance marker from the vector (abR, Figure 1). The resolution...This vector contains sacB, an chloramphenicol resistance cassette, and the R6K origin of replication to...replicated in the recipient, the only antibiotic resistant colonies isolated are those where the allelic ...crossovers, they are just spontaneous antibiotic resistant clones. But if they are blue or magenta, they ...hospital-associated Gram-negative bacteria become resistant to antibiotics! References Espah Borujeni, Amin...

  13. Plasmids 101: A Brief History of Plasmids and an Improved eBook!

    Type
    Blog Post
    Published
    Oct. 29, 2015, 2:30 p.m.
    ...treated a tetracycline resistant plasmid, pSC101, and a newly developed kanamycin resistant plasmid, pSC102,...EPISOME-MEDIATED TRANSFER OF DRUG RESISTANCE IN ENTEROBACTERIACEAE VIII: Six-Drug-Resistance R Factor.” Journal of...other extrachromosomal particles, particularly Resistance or R-factors. Like F-factors, R-factors could... selected for E. coli transformants that were resistant to both. When this proved successful, pSC101 became...Lederberg, Joshua. "Cell Genetics and Hereditary Symbiosis" Physiological Reviews 32.4 (1952) 403-430. Pubmed... PMC314833. Datta, Naomi. “Transmissible Drug Resistance in an Epidemic Strain of Salmonella Typhimurium...

  14. A Guide to Getting Started in Undergrad Research

    Type
    Blog Post
    Published
    June 8, 2021, 1:15 p.m.
    ...official thesis lab Undergraduate student Roles and responsibilities can vary "Lab assistants" usually...materials, lab maintenance Administrative assistant: assists PI with admin duties Staff scientist: similar...the lab Official title is typically Professor, Assistant Professor, Associate Professor Sometimes teaches...Graduate student Master's or PhD student Works on a thesis project to earn a degree "Rotation student" - some...usually wash glassware, prep media, etc. "Research assistants" are more involved in the science, experimental...independent project. Lab technician / Research assistant Typically has a Master's or Bachelor's degree..., as well as graduate students (your teaching assistants), about their experiences. Research requires ...

  15. Fluorescent Proteins 101: Visualizing Subcellular Structures & Organelles

    Type
    Blog Post
    Published
    June 22, 2017, 2:30 p.m.
    ... Open source image analysis software tools can be used for colocalization analysis. Some examples include...of quantitative colocalization analysis methods and useful image analysis software tools. Pitfalls, limitations...simply “looking at them”. Modern cameras and image analysis software are highly sensitive to detecting light..., and customized CellProfiler pipelines. Image analysis software produces quantifiable results and statistics...different experimental setups. A colocalization analysis example is given in Figure 2 (adapted from 4) ...Proteins. Ruusuvuori, Pekka, et al. "Quantitative analysis of dynamic association in live biological fluorescent...

  16. An Introduction to Adenovirus

    Type
    Blog Post
    Published
    Oct. 3, 2023, 1:15 p.m.
    ...vector that achieves persistent transgene expression in certain cell types (Tatsis et al., 2007), potentially...expression, and protein translation. Its genome consists of “early” (E) and “late” genes, and the transition...potentially because some of the wild-type viruses can persist as a latent infection in certain tissue reservoirs....1004821. PMID: 26042599. Pereira, H. G. (1972). Persistent infection by adenoviruses. J Clin Pathol Suppl...doi.org/10.1038/s41586-023-05949-1. PMID: 36996871. Tatsis, N., Fitzgerald, J. C., Reyes-Sandoval, A., Harris-McCoy...., & Ertl, H. C. J. (2007). Adenoviral vectors persist in vivo and maintain activated CD8+ T cells: implications...very late stages of infection for efficient cell lysis and release of adenovirus from infected cells. J...

  17. Using AAV for Neuronal Tracing

    Type
    Blog Post
    Published
    Aug. 9, 2018, 1:04 p.m.
    ...actin and myosin, and enzymes for transmitter synthesis. Retrograde transport is used for transport of...vivo, microinjection, pressure injection or iontophoresis (applying small electric currents via inserted...methods are naturally occurring viruses that infect, persist, and migrate within neurons, and that are also ... Not all measures of transport efficiency are consistent which may be caused by the diversity of techniques...are not fully understood and may be a form of transcytosis where the AAV particle is taken up on one side...cross the blood brain barrier by endothelial transcytosis (Foust et al., 2009; Merkel et al., 2017), and...Aschauer, D.F., Kreuz, S., and Rumpel, S. (2013). Analysis of transduction efficiency, tropism and axonal...

  18. Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

    Type
    Blog Post
    Published
    Nov. 1, 2018, 12:35 p.m.
    ...systems that are widely present in bacteria. They consist of a toxin which can affect a variety of cellular...division”). The encoded toxin-antitoxin system consists of CcdB (toxin) and CcdA (antitoxin) where CcdB...sequences in RNA resulting in a blockage of protein synthesis. The antitoxin MazE, which binds and inhibits ... contains the mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked...) contained within it by selecting for Zeocin resistance. Then, counter-selection with methanol medium...systems Because restriction modification systems consist of two enzymes that have opposing activities, it...the systems just described, the hok/sok systems consist of three components: Host killing (hok), a long-living...

  19. Plasmids 101: Control Plasmids

    Type
    Blog Post
    Published
    April 9, 2015, 3:29 p.m.
    ... in every experiment-based investigation of a hypothesis: proper controls! For every experiment, an investigator...ability of the cells to express the protein.   In analysis of the result in Figure 1, an internal control...includes the aforementioned internal control and is consistent with the result in Figure 1 could look like this...associated with the independent variable should be consistent over time and from different preparations of ...replicate control is important, it speaks to the consistency of the reagents and hands performing the experiment...experiment, not the reproducibility or consistency of the observed phenotype associated with the independent...replicates control for the reproducibility and consistency of the observed phenotype associated with the...

  20. Viral Vectors 101: Integrase-Deficient Lentiviral Vectors

    Type
    Blog Post
    Published
    May 13, 2025, 1:15 p.m.
    .... Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene...lentiviral vector: 1. No risk of insertional mutagenesis Lentiviral vectors tend to integrate into transcriptionally...2002), thus increasing the risk for insertional mutagenesis. This integration could lead to an undesirable...harbor these unwanted interruptions. Insertional mutagenesis is especially concerning in clinical settings...acute lymphoblastic leukemia due to insertional mutagenesis (Howe et al., 2008). 2. Transient expression ...cells, as they avoid the risk of insertional mutagenesis while still allowing for stable expression of...Sander, J. D. (2013). High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells...

  21. CRISPR 101: Engineering the Plant Genome Using CRISPR/Cas9

    Type
    Blog Post
    Published
    Oct. 11, 2016, 2:30 p.m.
    ... (2016) Enhanced Rice Blast Resistance by CRISPR/Cas9-Targeted Mutagenesis of the ERF Transcription Factor...longer shelf life and the development of disease-resistant crops to increase agricultural yield (Wang et ...including commonly used model organisms like Arabidopsis and Medicago truncatula and several crop species...sequence and ~75 nucleotide scaffold sequence) is consistent between plants and other organisms, but the promoter...GOI) into plant cells. A) The system typically consists of a Ti plasmid, into which the GOI is inserted...2013) Plant genome editing made easy: targeted mutagenesis in model and crop plants using the CRISPR/Cas...homoeoalleles in hexaploid bread wheat confers heritable resistance to powdery mildew. Nat Biotechnol 32:947–951 ...

  22. Plasmids 101: Positive and Negative Selection for Plasmid Cloning

    Type
    Blog Post
    Published
    Aug. 22, 2019, 12:43 p.m.
    ...antibiotic resistance cassette and you aren’t introducing an additional antibiotic resistance cassette ...where the inserted DNA includes an antibiotic resistance marker. After transformation, plate the cells...into the plasmid backbone contains a kanamycin resistance cassette, you would plate the cells on kanamycin-containing...cells carrying plasmids that contain a kanamycin resistance cassette will grow. However, often times, the...often used for cloning in yeast since antibiotic resistance spontaneously arises in yeast. For example, Markus...occurring genes for cloning purposes (antibiotic resistance genes, toxin genes, and the sacB gene). By tapping...

  23. Quantifying DNA? Here are Five DNA Quantification Methods to Consider

    Type
    Blog Post
    Published
    July 21, 2020, 1:15 p.m.
    ...salts. Capillary electrophoresis Quantifying DNA using capillary electrophoresis is similar to quantifying... using gel electrophoresis. Except, it’s smaller. And automated. Unlike gel electrophoresis, you only ... sample concentration for you. Agarose gel electrophoresis Though not the quickest way to quantify DNA...nanofluidic channel and the sample is separated by electrophoresis. Smaller fragments migrate quicker than larger...quantification of a RNA ladder using capillary electrophoresis which shows fluorescence units over time. Smaller...

  24. Using CRISPR/Cas9 to Edit Disease Out of the Genome

    Type
    Blog Post
    Published
    June 23, 2022, 1:15 p.m.
    ...through a phase of crisis where they 'collect' a number of mutations.” Cystic fibrosis may not be the most...Cas9 genome editing system to correct the cystic fibrosis transmembrane conductor receptor (CFTR) by homologous... intestinal stem cells of patients with cystic fibrosis (8).   The difference with CRISPR/Cas9 in comparison...stem cells isolated from two patients with cystic fibrosis. They were able to demonstrate functional correction...said Gerald Schwank, first author of the cystic fibrosis report. “This is a big advantage over induced ...Cas9 in Intestinal Stem Cell Organoids of Cystic Fibrosis Patients." Cell Stem Cell. 13, 653-658 (5 December...

  25. Viral Vectors 101: Viruses as Biological Tools

    Type
    Blog Post
    Published
    March 21, 2023, 1:15 p.m.
    ...breakdown of the nuclear envelope, which occurs during mitosis. The most well-known retrovirus is HIV. Lentiviruses...the fear of random genomic integration causing mutagenesis in the host. Adenoviruses can deliver relatively...Dividing Cells Moderate Immunogenicity Insertional Mutagenesis Possible Lentivirus Infects non-dividing...non-dividing cells Insertional Mutagenesis Possible Adenovirus Remains Episomal (Little Risk of...of Insertional Mutagenesis) High Gene Expression Infects Non-Dividing Cells Large Packaging Capacity (... AAV Remains Episomal (Little Risk of Mutagenesis) Very Low Immunogenicity Infects Non-Dividing...Integration of murine leukemia virus DNA depends on mitosis.” EMBO J. 12 (1993) 2099-2108. PubMed PMID: 8491198...

  26. Expanding the Targeting Scope and Editing Efficiency of Adenine Base Editors

    Type
    Blog Post
    Published
    March 17, 2020, 1:32 p.m.
    ...lab developed the Phage-Assisted Continuous Evolution (PACE) and Phage-Assisted Non-Continuous Evolution...Base editors make specific DNA base changes and consist of a catalytically impaired Cas protein (dCas or... to base editor activity and editing speed and consists of the following components: A plasmid in the.... A phage encoding the deaminase TadA-7.10 A mutagenesis plasmid controlling the phage mutation rate ...stringent phage selection. Figure 2: Phage-assisted evolution of base-editing activity. A plasmid-...Zeng J, Bauer DE, Doudna JA, Liu DR (2020) Phage-assisted evolution of an adenine base editor with improved...

  27. Deep Dive: Statistical Tests (Comparisons)

    Type
    Blog Post
    Published
    March 7, 2023, 2:15 p.m.
    ...the heavy lifting, so don’t let the statistical analysis of your data get you hung up. Here we will dive... for you. Statistics – The basics Statistical analysis is what determines if there is statistical significance...account for subject-to-subject variation in its analysis (the test assesses the difference of the group...differences exist between. For example, if an ANOVA analysis on a five-sample data set yields statistical significance...pairings, additional tests, referred to as post-hoc analysis, must be performed. These will be discussed later...information. A post hoc test, done after your initial analysis can provide information on specifically which ...statistically different within your ANOVA/ANCOVA analysis. If you have performed a MANOVA/MANCOVA and want...

  28. Important Considerations When Using AAVs

    Type
    Blog Post
    Published
    June 13, 2017, 2:30 p.m.
    ...insertional mutagenesis in humans (1) and AAVs are non-enveloped viruses, so they are resistant to alcohol-based... viruses, AAVs normally require second strand synthesis before gene expression begins. ScAAV avoid this... Russell, David W. "AAV vectors, insertional mutagenesis, and cancer." Molecular Therapy 15.10 (2007):...PMID: 17882145. 2. Zincarelli, Carmela, et al. "Analysis of AAV serotypes 1–9 mediated gene expression ...Dominik F., Sebastian Kreuz, and Simon Rumpel. "Analysis of transduction efficiency, tropism and axonal...promote efficient transduction independently of DNA synthesis." Gene therapy 8.16 (2001): 1248. PubMed PMID:...8. Ferrari, Forrest K., et al. "Second-strand synthesis is a rate-limiting step for efficient transduction...

  29. Sequencing Options for CRISPR Genotyping

    Type
    Blog Post
    Published
    Oct. 4, 2016, 2:30 p.m.
    ...off-target analysis in order to meet clinical approval. It is key to maintain a consistent set of standards...connections can help alleviate the reproducibility crisis in biology. It is key to address these concerns... The results are then visualized using gel electrophoresis. Surveyor™ and T7E1 have been widely adopted...including Digenome-seq (Kim et al. 2015) for in vitro analysis, IDLV for in vivo detection (Gabriel et al. 2011..., Yanfang, et al. "High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells... sequencing, pyrosequencing, and melting curve analysis for the detection of KRAS mutations: diagnostic...Gabriel, Richard, et al. "An unbiased genome-wide analysis of zinc-finger nuclease specificity." Nature biotechnology...

  30. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    Published
    July 20, 2021, 1:15 p.m.
    ...cytometry provides analysis at the cellular level, it cannot be used for subcellular analysis such as morphology...lymphocytes and “gate” that population for further analysis. Since different populations of immune cells express...around their desired cell population for further analysis. In this example, the lymphocyte population is...Include proper controls To ensure accurate data analysis, include a complete set of controls. Ideally, ...allow you to omit dead or dying cells from your analysis; dead or dying cells tend to have more autofluorescence... panel will help you set up gating during data analysis.  We hope that this blog has provided you with... an overview of flow cytometry for single cell analysis and some useful background information for setting...

  31. Hot Plasmids February 2024

    Type
    Blog Post
    Published
    Feb. 20, 2024, 2:45 p.m.
    ...Here's what you'll find in this post: Peptide-assisted genome editing for primary cells Monomeric StayGold...Recombinant anti-FLAG tag antibodies Peptide-assisted genome editing for primary cells By Susanna Stroik...optimizing the Cas protein delivery with Peptide-Assisted Genome Editing (PAGE). They fused the Cas enzyme...cell-penetrating peptides and co-incubate it with an “assist peptide” that facilitates endosomal escape. Combining...mCherry reporter (mChe), with co-incubation with assist peptide (AP). C) Workflow for Cas-RNP-PAGE editing...primary T cells, by co-incubation of Cas-RNP and assist peptide. Adapted from Zhang et al. 2023 with permission...of human and mouse primary cells using peptide-assisted genome editing. Nature Biotechnology, 10.1038/...

  32. Polymerase Chain Reaction Overview and Applications

    Type
    Blog Post
    Published
    Aug. 10, 2021, 1:15 p.m.
    ...heating and cooling steps. PCR relies on heat resistant DNA polymerase from the thermophilic bacterium...aquaticus (Taq). Taq polymerase is thus a heat resistant enzyme that can withstand changes in temperature...generating mutated sequences for site-directed mutagenesis.  Quantitative polymerase chain reaction, qPCR... high throughput SNP genotyping, gene deletion analysis, and RNA detection.  A summary of the types of...interest  Molecular Cloning Site-directed mutagenesis Molecular Diagnostics qPCR Quantify starting...pathogen identification SNP genotyping Gene deletion Analysis RNA detection     References and Resources... PCR  Plasmids 101: Colony PCR Site Directed Mutagenesis by PCR Resources on Addgene.org Protocol: Polymerase...

  33. CRISPR 101: Any Base Transversion Editors

    Type
    Blog Post
    Published
    April 1, 2025, 1:15 p.m.
    ...newly-deaminated inosines. Pro tip! The nucleoside inosine consists of the nucleobase hypoxanthine + a ribose sugar...this promising start, the team used rational mutagenesis to produce the adenine transversion editor AYBEv3...editing, they co-expressed Polη, a translesion synthesis polymerase known to preferentially incorporate...and ABEs, they could be useful for saturation mutagenesis, and they provide a strong proof of concept for... editor (gGBE) (Tong, Liu, et al., 2023). gGBE consists only of engineered MPG fused to nCas9. First, ...empty space. Over several rounds of rational mutagenesis, the team progressed to gGBEv6.3, which was capable...., & Chang, X. (2024). Protein language models-assisted optimization of a uracil-N-glycosylase variant...

  34. Hot Plasmids: Fall 2024

    Type
    Blog Post
    Published
    Nov. 21, 2024, 1:30 p.m.
    ...expression levels comparable to GFP during early embryogenesis (Figure 3). mScarlet-I3 also offered clearer... Pe K., Cesar M., Hobert O. (2024) Comparative analysis of new mScarlet-based red fluorescent tags in ... common human pathogen notorious for its high resistance to antibiotics. Several CRISPR/Cas-based systems...abscessus’ high rate of spontaneous antibiotic resistance interfering with selection. Using an inducible...understanding of its biology, pathogenicity, and resistance mechanisms.     Figure 4. Overview of...plasmid containing tetracycline repressor TetR and resistance selection marker KanR. This strain is then transformed... cassette on a plasmid expressing mCherry and resistance selection marker HygR. The CRISPR system is induced...

  35. To Codon Optimize or Not: That is the Question

    Type
    Blog Post
    Published
    Nov. 12, 2020, 2:15 p.m.
    ...codon usage important? Translation and protein synthesis relies on transfer RNAs (tRNAs). tRNAs bind to...2010). Figure 1: An overview of peptide synthesis. A ribosome interacts with mRNA and charged tRNAs... altering the sequence of the protein. As DNA synthesis has become quite cost-effective it seems easy ...algorithms/resources (Athey et al., 2017) and many DNA synthesis companies, such as IDT, Genewiz and GenScript,...system of choice. ThermoFischers GeneArt Gene Synthesis NovoPro Labs Codon Optimization Tool (ExoOptimizer... Hausser J, Syed AP, Bilen B, Zavolan M (2013) Analysis of CDS-located miRNA target sites suggests that....139758.112 Mauro VP, Chappell SA (2014) A critical analysis of codon optimization in human therapeutics. Trends...

  36. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    Published
    Sept. 17, 2024, 1:15 p.m.
    ...phosphatase inhibitors to your lysis buffer. Additionally, protein lysis should be done at 4 °C or on ice...conditions Adjust lysis conditions: time, temperature, and/or buffer to increase lysis Add sonication...temperature. Some proteins aggregate at 95 °C, a common lysis temperature, so consider a longer incubation (10...ladder on the lefthand side. Whatever you do, be consistent and document it.   Did it work? Save yourself...chemical buffer around, which works well and consistently, to help troubleshoot background issues. It’...sonication step to protein lysis Splice variants Check literature Posttranslational modifications ...Run control protein (if available) and note in analysis Plasmid-based proteins: plasmid sequence not...

  37. Targeting HIV-1 with CRISPR: Shock and Kill or Cut it Out?

    Type
    Blog Post
    Published
    May 10, 2016, 2:30 p.m.
    ...cell cycle progression, or apoptosis with Cas9/gRNA expression. A pooled analysis found no evidence of Cas9... HIV-1 toxic proteins, CRISPR/Cas9 SAM caused apoptosis, indicating a true “shock and kill” response. ...binding. It’s also possible that HIV-1 may evolve resistance to CRISPR therapies through PAM or seed sequence...multiple drugs to lower the odds of developing resistance. Shortly after Kaminski et al. published their...not ablate viral function, but rather promote resistance to CRISPR/Cas9. While these results are discouraging...synergistic activation mediator (SAM) induces specific, persistent and robust reactivation of the HIV-1 latent reservoirs...

  38. Evolution of Brainbow: Using Cre-lox for Multicolor Labeling of Neurons

    Type
    Blog Post
    Published
    April 24, 2015, 2:39 p.m.
    ...generate additional recombination events, and the synthesis of alternative loxP sites to specify distinct ...schematic at the bottom of this post for a basic synopsis of the differences amongst the Brainbow systems...neurons’ somata. Second, the system did not permit analysis by immunostaining. Although the fluorophores used...cross-reactivity, opening the door for immunostaining analysis. To achieve even cell labeling, they generated.... Epub 2008 Apr 30. PubMed. Intestinal crypt homeostasis results from neutral competition between symmetrically...genetic multicolor cell labeling for neural circuit analysis in Drosophila melanogaster. Hadjieconomou D, Rotkopf...multispectral cell labeling for cell tracing and lineage analysis in zebrafish. Pan YA, Freundlich T, Weissman TA...

  39. What's New in CRISPR - September 2019

    Type
    Blog Post
    Published
    Sept. 10, 2019, 12:56 p.m.
    ...genes chosen from an analysis of human metastasis cohorts and a prior mouse metastasis CRISPR screen. The...discovered a new Cas protein, CasX, from metagenomic analysis of microbial DNA from groundwater. Now, they’ve...knockout libraries for cancer research. The Mouse Metastasis CRISPR-Cpf1(Cas12a) Double Knockout Library is...tumor suppressor genes (TSG). Find the Mouse Metastasis CRISPR-Cpf1 Double Knockout Library Find the...

  40. pSiM24: Simplifying Plant Genetic Engineering

    Type
    Blog Post
    Published
    Sept. 29, 2015, 2:30 p.m.
    ...continue to improve plant nutrition, yield, and pest resistance. Much of plant genetic engineering revolves around...functions in multiple species, including tobacco and Arabidopsis. Improving Agrobacterium binary vectors Vectors...domains and characterized in studies of tobacco, Arabidopsis, and corn. For both β-glucuronidase (GUS) and...efficiently inherited in both transgenic tobacco and Arabidopsis. Finally, Sahoo et al. demonstrate that this ...variety of plant species. The kanamycin antibiotic resistance gene is controlled by a weaker constitutive Nos...Plant Synthetic Biology Quick, Versatile Plant Transgenesis with GreenGate Plasmids Interview: Nicola Patron...
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