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Showing: 241 - 260 of 960 results

  1. CRISPR 101: Making a Knock-In Cell Line

    Type
    Blog Post
    ...location. HDR depends on resection at the break site to form ssDNA and uses the ends of the ssDNA to find a repair...increase HDR frequency by committing a break to performing HR before other factors were loaded onto the ...

  2. Antibodies 101: Immunoprecipitation

    Type
    Blog Post
    ...antibody-target protein immune complexes are allowed to form. Following incubation, beads are used to immobilize...-40 or triton-X, to preserve native protein conformations and interactions. For difficult to release proteins... in the lysis buffer, keep samples on ice, and perform the IP at 4oC. When choosing the capture antibody... recognizes the target protein in its native conformation. Frequently, the antibodies used for a standard... are completely removed. Wash steps should be performed even with a pre-cleared sample.  Eluting the target...and negative controls for an IP. When possible, perform the IP in parallel with both a sample known to ...order to identify any non-specific interactions, perform parallel IPs with beads only and an isotype control...

  3. Don’t FRET: Bimolecular Fluorescence Complementation Makes Visualizing Protein-Protein Interactions Easy

    Type
    Blog Post
    ...protein interaction partners (PIPs), the FP fragments form the complete FP because the PIPs they’re tethered...to bring them into close proximity (Figure 1). Formation of the complete FP results in a fluorescent signal...selection of FP fragments and, preferably, some information about the structures of the tested PIPs. Many...fuse the FP fragments. In the absence of this information, you can fuse each FP fragment to the N- and ...your results. Unfortunately, without structural information, constructing appropriate controls may requires...

  4. Hot Plasmids - August 2020

    Type
    Blog Post
    ...synthetic oscillator, bistable network, stripe pattern-forming incoherent feed-forward loop. Find the plasmids...systems at Addgene! Schematic of the evolution platform to generate TEV protease variants. Catalysis by...the right places, which can provide misleading information on where exactly a structure or protein is located...dissolved at a higher concentration, and results in uniform NB biotinylation. Once induced, the NBs could be...developed fluorescently labeled piggyBac vectors for uniform and sustained expression of single or multiplexed...

  5. Multiplexed Capture of Promoter-enhancer 3D Chromatin Structures Using CRISPR

    Type
    Blog Post
    ...chromatin, the complex of DNA and proteins that forms chromosomes. These long range DNA interactions are... are often detected with chromosome conformation capture (3C) based methods, such as 5C or Hi-C (Han et...generated with current 3C methods provide useful information about chromatin interactions, they are grainy...for answering more questions about chromatin conformations. The Xu lab presented three ways to use CAPTURE... capture of chromatin interactions The Xu lab performed a proof-of-concept experiment to show that CAPTURE...original CAPTURE method. Both CAPTURE methods outperformed other 3D chromatin methods such as ChIA-PET ...is detection of temporal changes in chromatin conformation, like those that occur during development and...

  6. Fluorescent Proteins 101: Fluorescent Protein Timers

    Type
    Blog Post
    ...changes in gene expression as new body sections are formed. In synthetic biology, FP timers might be able ...isolate particular organelles and their contents, or perform in situ hybridization experiments. In many cases...like the patterning of nascent tissues and the formation of limbs are correlated with changes in gene expression...and developmental timer of neural fiber bundle formation." Journal of Biological Chemistry 276.32 (2001...

  7. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    .... For instance, the fluorophore and quencher may form the base of the stem in an RNA stem-loop structure...and detection - since they frequently undergo conformational changes after target binding, aptamers are ...fluorescence-activated cell sorting (FACS) of E. coli transformed with aptamer libraries, the Jaffrey group selected...

  8. Designing Your Chalk Talk for the Academic Job Interview

    Type
    Blog Post
    ...vision. Practice drawing and writing everything in a form that is easily visible from the back of the room... vision. Organize your vision into a chalk talk format. Practice. Define your research vision A chalk...find, and how you will interpret and use the information. In contrast, a list of experiments or a laundry...Organize your research vision into the chalk talk format Once you have developed your research vision, you...most of your limited speaking time. Beyond the information you seek to convey in your chalk talk, there'...

  9. Tips for a 1st time AAV user (by a Rookie AAV user)

    Type
    Blog Post
    ...are required for proper packaging of AAV, but also form secondary structures that are prone to deletion ...and XmaI restriction sites). Another way is to transform AAV transfer plasmids into bacterial strains, ...found in the culture media, you should consider performing PEG precipitation. Instead of sonicating the ...

  10. Plasmids 101: Codon usage bias

    Type
    Blog Post
    ...choice, you should perform a functional assay to make sure your protein isn’t forming inclusion bodies ...

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

    Type
    Blog Post
    ... in spacer acquisition following infection, as a form of “bait” to identify new CRISPR-associated proteins...target binding and cleavage through a series of conformational changes in the Cas13a molecule. Like Cas9, ...

  12. New Tools Enable CRISPRa for Neuroscience Applications

    Type
    Blog Post
    ...specify and assemble diverse arrays of neurons, forming the neuronal circuits that will later be refined...traditional cDNA overexpression: isoform-specific gene expression. Gene isoforms refer to mRNAs generated from...untranslated or coding regions. Typically, each gene isoform has a distinct transcriptional start site. Because...transcriptional start sites in order to better understand isoform-specific gene regulation — at least, more straightforward...straightforward than generating cDNA for each gene isoform individually. To highlight this advantage, they...neurons and synapses as well as long-term memory formation. The Bdnf gene possesses 9 distinct transcriptional...with CRISPRa, the Day lab singled out two Bdnf isoforms (I and IV) for investigation. Co-transduction ...

  13. Negative Can Be Positive: Open AAV Data with Addgene

    Type
    Blog Post
    ...highlights the dearth of AAV vector information provided in openly accessible forms like publications. Why not ... two years testing and reformulating our AAV construct to optimize performance in our system and yet none...literature, and as a result can leave scientists under-informed with respect to how different serotypes, promoters...this one set of experiments didn’t provide any information on other important factors such as what is the...adjacent areas impacted the experiments we wanted to perform. It required a great deal of time and effort to...packaging viral particles (6), can influence vector performance. Open science initiatives, like Addgene’s Data...negative data and instead focus on getting this information out into the public where it can be utilized ...

  14. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

    Type
    Blog Post
    ...compaction—on the order of 700 terabytes per gram—and form the X-shaped chromosomes we all know and love. To...enhancers and genome math come in. They help us to transform raw genomic coordinates into meaningful experiments...tri_methylation In the next section we’ll apply the above formula to a real-world experiment. We’ll start with ChIP-seq...

  15. Minigenomes - a Safe Way to Study Dangerous Viruses Like the Ebola Virus

    Type
    Blog Post
    ... associates with the viral polymerase, which is formed by a complex of two proteins: the large (L) catalytic...measured to quantify (indirectly) transcription performed by the viral polymerase. Alternatively, the mRNA...independently. In addition, experiments can be performed in the absence of some viral proteins (compared... minigenome plasmid per well (in a 6 well plate format). However, since plasmid quality and transfection...

  16. Avoiding the Dark Side of Fluorescent Protein Fusions with mOX FPs

    Type
    Blog Post
    ...and others have reported that FP cysteine residues form inappropriate disulfide bonds in the secretory pathway...modifications including glycosylation, disulfide bond formation, and proteolytic cleavage. FPs localized to organelles...fusion protein localization. For example, the formation of inappropriate inter-chain disulfide bonds between...because of the correct folding and fluorophore formation by most, if not all, of the FP fusions. Our moxFPs...

  17. Tips from the Repository Trenches: Using Barcodes to Track Samples

    Type
    Blog Post
    ...Barcoded tubes come in many forms. The 2D barcode can store a lot of information in a small space and is ... treated to easily take in plasmids (transformation). Transformed bacteria are then grown up on antibiotics...received is immediately transformed and stored. Some samples need to be transformed with a micropulser using... (98.2%) transform the first time and can be stored right away. Every week, the lab performs an average...average of 140 transformations. The lab typically transforms newly deposited samples from Monday through Wednesday...delivered by a mail courier and then the journey of transformation and storage begins. Some samples are submitted...barcoded tubes that are scanned into our Laboratory Information Management System (LIMS), a database we use to...

  18. Plasmids 101: Protein tags

    Type
    Blog Post
    ...can also protect against proteolysis. That’s one form of instability. Prokaryotes can also have a hard...Thioredoxin A (TrxA) that improve disulfide bond formation in order to help keep your protein soluble.  ... better assess their function. More detailed information and some protocols can be found in the references...

  19. Negotiating Work and Life: How to Find the Joy

    Type
    Blog Post
    ... local family to help, create “fake” family by forming a social circle of people in the same situation... and/or your spouse won’t be at every school performance, chaperone every field trip or cheer at every...
Showing: 241 - 260 of 960 results