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Showing: 101 - 120 of 710 results

  1. Transferable Skills Guide: Cross-team Communication

    Type
    Blog Post
    Published
    Nov. 21, 2019, 1:53 p.m.
    ...“no.” When there was room for misinterpretation, that misinterpretation was definitely happening. Our ...terminology of a new field. Annotate: When creating important documentation, including SOPs or project-...were leaving these meetings with different interpretations of what we discussed and decided. I needed ...definitions in our lexicon. We created documents and presentations, gave real life examples of when and how to ...frequently used words like this when starting rotations or my postdoc would have simplified the first ... ambiguous terms. Defining words and applying annotation can be particularly useful in academic labs when...

  2. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

    Type
    Blog Post
    Published
    Dec. 15, 2016, 3:57 p.m.
    ... screen plasmids for the appropriate mutations.  Point mutations and other subtle changes should be confirmed...molecular cloning; however, one of its biggest limitations is that sequence modifications can only be made...selectable and non-selectable sequences, point mutations or other small base pair changes, and the addition...nucleotides, while ssDNA substrate is best for point mutations or changes of only a few base pairs.  dsDNA Substrate...the two options, but these cells are prone to mutations and will have more unintended changes to their...Taq polymerase to generate insert ssDNA Point mutations or changing a few nucleotides 0.1-1% or 25-50%...effects of Gam expression and to limit spontaneous mutations that occur when Red is constitutively expressed...

  3. Pathways Over Time Plasmids Engage Students in Functional Genomics Research

    Type
    Blog Post
    Published
    May 22, 2018, 1:38 p.m.
    ...projects in introductory labs. Genome projects use computational methods to identify genes by their similarities...proteins have acquired divergent functions due to mutations accumulated over time? Undergraduates can help...inexpensive. In addition, a complete set of well-annotated deletion strains is available (6). Methionine ...ura3 mutants and cannot grow without uracil supplementation (6). Expression of MET/Met genes from the pYES2.1...blotting in other experiments (Figure 2).   Complementation reveals conservation of function Students have...Addgene, to successfully analyze Met protein complementation. In the procedure diagrammed in Figure 3, students...promoter controls MET/Met gene expression, complementation should only occur when galactose replaces glucose...

  4. Split-BioID: An Improved Method for Studying Protein-Protein Interactions

    Type
    Blog Post
    Published
    May 1, 2018, 1:57 p.m.
    ...type of experiment is called a protein fragmentation complementation assay (PCA). In the Split-BioID method...FRET is another example of a protein fragmentation complementation assay that can be used to study binary...individual protein. The Bethune Lab has overcome this limitation by creating Split-BioID, a spatiotemporally controllable...Protein Interactions. Split-BioID is a protein complementation assay where BirA*, an E. coli biotin ligase...proteins. One caveat of Split-BioID is that the orientation of the BirA* fusion matters! Schopp et al saw...interact with the bait proteins. Another potential limitation of Split-BioID is its speed of labeling. BioID...

  5. Early Career Researcher Toolbox: Finding Relevant Papers

    Type
    Blog Post
    Published
    Jan. 9, 2020, 1:45 p.m.
    ...number of citations highly influenced by this paper. Influence is measured by the number of citations and the...extract important features such as influential citations, images, and key phrases. It also takes into account... account how papers are connected via citations, in order to return results that are more relevant to ...with greater influence, the average number of citations per year and the number of other manuscripts that... When you view a paper’s entry, the number of citations that paper has is displayed at the top of the ... the context of these citations. The number of manuscripts that cite the background, methods, or results...all publications by an author, total number of citations, and the number of publications that heavily cite...

  6. Polymerase Chain Reaction Overview and Applications

    Type
    Blog Post
    Published
    Aug. 10, 2021, 1:15 p.m.
    ..., or for generating mutated sequences for site-directed mutagenesis.  Quantitative polymerase chain reaction...genotyping. It’s extremely useful but is not as quantitative as other methods of PCR. Theoretically scientists...reaction, qPCR Quantitative polymerase chain reaction (qPCR) also known as real time PCR is a PCR technique...material in your sample.  qPCR can also be used to quantitate RNA levels using reverse transcription-qPCR (...sequences that are rare, for example, rare alleles or mutations. ddPCR also does not require a reference or standard...is determined and then analyzed using Poisson statistics to determine the concentration of the original...PCRs that use a single pair of primers, like those stated above. Multiplex PCR is also used in high throughput...

  7. Plasmids 101: How to Name Your Plasmid in 3 Easy Steps

    Type
    Blog Post
    Published
    Feb. 13, 2014, 1 p.m.
    ...contains a mutation or modification, this should be included in the plasmid name. Mutations are generally...abbreviation of the mutated amino acid currently at that position. In contrast, unmutated or Wild Type (wt...your plasmid name. This is often a 3-6 letter representation of the gene (or DNA sequence). pBACKBONE-hGene...change. The proper way to denote an amino acid mutation is to list the one letter abbreviation of the ...   Example: If the Glutamine at position 295 was mutated to an Alanine, Q295A.  Mutant version: pBACKBONE-Flag-hGene...

  8. Make a Splash: Notions of Scientific Impact Are Evolving

    Type
    Blog Post
    Published
    Dec. 5, 2014, 2:54 p.m.
    ...Considering citations to all articles, Anurag Acharya and colleagues report, the percentage of citations to articles...to 1975 and is based on the average number of citations for recent articles. Of course, a publication ...measure scientific impact. Even if we stick to citation counts for a moment, it’s clear that articles ...articles in non-elite journals went from 27% of all citations in 1995 to 47% in 2013 – yes, almost half.   The...Accessibility is surely also behind the increase in citations for older publications. (As an interesting aside...the amount of protein in a solution.) But are citations and journal publications really the best way to...

  9. SciComm: Taking Science to Elementary and Middle Schools

    Type
    Blog Post
    Published
    July 9, 2019, 12:52 p.m.
    ...parents looking for experts to come give guest presentations. If you’re a scientist thinking about reaching...already discussed the idea of my giving a CRISPR presentation to their students. I didn’t think of this opportunity...postdocs, and other working scientists, creating a presentation for 8th graders was a little intimidating. Too...received from the teachers, I formulated the presentation as follows (which could be adapted for any topic...do? I spent more time on this section of the presentation because, in my opinion, it was more important...teachers with whom I worked, for feedback on my presentation, she said “it was such a valuable extension ... of CRISPR. Two of the examples I used in my presentation had only become public in the previous two weeks...

  10. Isolating Genomic Regions of Interest with the CRISPR System

    Type
    Blog Post
    Published
    May 30, 2019, 12:44 p.m.
    ... DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) because any engineered DNA-binding...region of the transcription factor IRF-1. Immunoprecipitation, using an antibody targeting the 3xFLAG tag...purification of a specific genomic region, numerous adaptations of this system have been made in different fields...also has great potential for improvements and adaptations that can provide an increased understanding of... DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR." Biochemical and biophysical... DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)." Scientific reports 3 (2013): 3171...and Hodaka Fujii. "Insertional chromatin immunoprecipitation: a method for isolating specific genomic ...

  11. The Effect of COVID-19 on Liu Lab Plasmid Requests From Addgene Data

    Type
    Blog Post
    Published
    July 16, 2020, 1:15 p.m.
    ...COVID-19 cases each week. Statistical Modeling Note -- If statistical modeling isn’t up your alley,... estimated total of 380,000 deaths worldwide (statistics provided by Google on 6/4/20). Many countries... open-ended question is difficult to answer quantitatively without broad, unbiased data. Therefore, we...order several plasmids at once, breaking the statistical independence assumption of the Poisson process...process. This modification also eases the interpretation of the parameters we infer, since scientific productivity...remind the reader that this is not a causal interpretation -- the effect would generally not be the same...His research uses applied machine learning and statistical methods for fundamental scientific discovery ...

  12. Interview: Hodaka Fujii on enChIP, New CRISPR Tools, and More

    Type
    Blog Post
    Published
    Dec. 2, 2014, 7:23 p.m.
    ... an invitation to join the Advisory Board of Addgene, I accepted the offer without any hesitation. I hope...transcription by using locus-specific chromatin immunoprecipitation (locus-specific ChIP). These methods consist...consist of insertional chromatin immunoprecipitation (iChIP) and engineered DNA-binding molecule-mediated...molecule-mediated chromatin immunoprecipitation (enChIP), both developed in the lab. In June 2014, Dr. Fujii joined ..., we developed the insertional chromatin immunoprecipitation (ChIP) (iChIP) technology, which uses locus-tagging... DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP). Fujita T, Asano Y, Ohtsuka J, Takada... DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) using CRISPR. Fujita T, Fujii H....

  13. Starter Guide to induced Pluripotent Stem Cells (iPSCs) Part 2:  Reprogramming and Transdifferentiation

    Type
    Blog Post
    Published
    Nov. 6, 2018, 1:12 p.m.
    ...differentiated somatic cell reverts back to a pluripotent state, which then proliferates and is redifferentiated...differentiated cell type to an undifferentiated state and then redifferentiation into the cell type of...Conversion of adult somatic cells into the pluripotent state. Redifferentiation - Conversion of the pluripotent...Dedifferentiation to an intermediate pluripotent state is not always obligatory in cell conversion processes...back to their most primitive pluripotent stem cell state, through transdifferentiation adult somatic cells...factors) More Chances of accumulation of harmful mutations More, due to selection from more cycles of proliferation...objective is to fix a disease-inducing genetic mutation in a patient, trying to transdifferentiate any...

  14. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    Published
    July 13, 2023, 1:15 p.m.
    ...such a small package. But don’t let the simple presentation deceive you; pooled libraries can be quite challenging...verified through NGS sequencing to confirm library representation. Transfection of the DNA pooled library and...transduction of cells, resulting in cell population mutated by the viral pooled library. These cells can then...protocol that amplifies without disrupting plasmid representation. A good place to start is with a protocol for...library by next-generation sequencing (NGS). The representation of your amplified library should be very similar...page, you can find protocols for sequencing, representative NGS results (read counts or another visualization...library and verify that you have not skewed the representation of your pooled library during the amplification...

  15. CRISPR 101: Targeting RNA with Cas13a (C2c2)

    Type
    Blog Post
    Published
    Sept. 21, 2017, 2:07 p.m.
    ... an enzymatically “active” state rather than reverting to an inactive state like Cas9 or Cpf1. As a result...proteins like SaCas9 and Cpf1 can circumvent the limitations associated with SpCas9. Cas13a (previously referred... HEPN domains in other proteins. As with Cas9, mutating key residues in the Cas13a molecule results in...system, Cas9 uses both a tracrRNA and crRNA to facilitate binding and cleavage of target DNA respectively...molecule through a uracil-rich stem loop and facilitates target binding and cleavage through a series ... to activate programmed cell death or a dormant state for bacterial cells that have been infected with...Zika virus, genotype human DNA, identify tumor mutations within cell-free genomic DNA, and detect COVID...

  16. Antibodies 101: Normalization and Loading Controls for Western Blots

    Type
    Blog Post
    Published
    April 8, 2025, 1:15 p.m.
    ..., I promise it isn’t.) The intuitive, qualitative interpretation of this gel is misleading. We can see...causes misleading qualitative conclusions, and it’s also problematic for computational quantification. ...experimental parameters. This is a job for a semi-quantitative western blot. Most western blots benefit from...Loading controls are even more important for semi-quantitative western blots. No pipetter is perfect, and proteins...lysate (10–40 µg) probed with β-actin (1:2000). Representative of three independent blots. B–C) Graphs showing...high-abundance single-protein controls in semi-quantitative immunoblotting. Journal of Neuroscience Methods...

  17. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    Published
    Dec. 6, 2022, 2:15 p.m.
    ....1c02201. LiLac sensor for lactate Stop and smell the flowers - or rather, the lactate! The latest biosensor...for lactate developed using a high-throughput multiparameter screen (Koveal et al. 2022). Lactate is a...previous lactate biosensors, and its affinity is better-matched to cellular concentrations of lactate. The...ZAP70 (ZtSH2) which binds only the phosphorylated state of an immunoreceptor tyrosinase-based activation...around 70 mM and 100 mM, respectively. Introducing mutations and using potassium-binding protein homologues...

  18. Plasmids 101: Antibiotic Resistance Genes

    Type
    Blog Post
    Published
    Jan. 30, 2014, 3:29 p.m.
    ...bactericidal) or slow growth/prevent cell division (bacteriostatic); however, the distinction between the two ...categories may be a bit of a gray area as some bacteriostatic reagents can kill bacteria when used at high...subunit; inhibits aminoacyl translocation Bacteriostatic 50-100 ug/mL in EtOH Polymyxin B polypeptide...inhibits protein synthesis (elongation step) Bacteriostatic 10 ug/mL Chloramphenicol   Binds 50S ribosomal...ribosomal subunit; inhibits peptidyl translocation Bacteriostatic 5-25 ug/mL in EtOH *In prokaryotes.     ...inactivates the gene and acts as a marker for the mutation. When designing these types of experiments it ...not to use the same resistance cassette for the mutation and for plasmid selection. Additionally, scientists...

  19. Technique: Probe Phage Genomes for Host Binding Proteins

    Type
    Blog Post
    Published
    Sept. 29, 2016, 2:30 p.m.
    ...DNA with phi29 polymerase prior to fragmentation can greatly facilitate downstream manipulations. Phi29 ...activity without the need for genome sequencing or annotation (5). In the event that genome sequencing can ...to enzymatic digestion, however, a physical fragmentation strategy is preferable. Nebulization involves...associated with cloning large fragments. After fragmentation, repair sheared ends with T4 DNA polymerase ...gently down onto a plate of colonies. Mark the orientation of each membrane on its plate before moving on...your membrane to several freeze/thaw cycles to facilitate cell lysis: alternating between 5 min at -20°...sequence the inserted DNA and characterize your new putative host cell-binding proteins! Many thanks to our...

  20. Building Global Connections with the International Mentorship Program USA-EUROPE

    Type
    Blog Post
    Published
    Nov. 6, 2015, 3:30 p.m.
    ...professionals has made possible a significant qualitative and quantitative expansion in the 2015-2016 academic ...does research at UK in the same field that I do: prostate cancer. She has guided me in my next steps. For...baseline self-assessment survey to determine their expectations of the program and their baseline skills. Mentors... as mentors to Spanish mentees, mentors also facilitated the development of bidirectional USA-Europe professional... pre-program self-assessment survey defining expectations of the program and their baseline skills and...improvements in writing fellowships, giving oral presentations, and preparing for job interviews, expanded ...Spanish scientific community overseas. Official representatives from different Spanish universities and high...
Showing: 101 - 120 of 710 results