...reprogramming efficiency through the expression of a shRNA against p53, a modified reprogramming factor cocktail, ...are preferred. This is to avoid the chances of insertional mutagenesis, which is the integration of transgenes...disruption of functional genes at the site of insertion and which can potentially promote tumorigenesis... been recorded in past gene therapy trials and remains a safety concern in the clinical field [4]. Three...Antigen-1 (oriP-EBNA-1) system where each vector contains a viral origin of DNA replication, OriP and the... pCXLE-hOCT3/4-shp53-F 27077 OCT3/4 and shRNA against p53 pCXWB-EBNA1 37624 transient EBNA-1   ...Road Ahead." Journal of Alzheimer's disease & Parkinsonism 6.5 (2016). PubMed PMID: 27882265. PubMed Central...

... apply for the position. This post on Business Insider nicely summarizes good job fair practice. Career...get online and take a look at the company or institution’s career page, where you’re likely to find current...conference or job fair, or it may be someone at your institution’s career center. Alternatively, it might even ...other graduate students and post-docs from your institution have gone, and try sending them a professional... professional development, not count on your institution, program, or adviser to do it for you. Expose...if one exists. Get online! Review company and institution career pages. Make yourself visible on LinkedIn... Reading and Outside Resources http://www.businessinsider.com/how-to-get-the-most-out-of-job-fairs-2014...

...Singletons in their bins.  The next component of Binning Singletons is when we bring all the bins together at ...invaluable to me as a bench scientist working at an institution where training opportunities weren’t always available...their own labs.  Fried chicken with a side of inspiration  I look back at the meeting with such fondness...for DNA that group similar sequences together in bins, we thought that binning Singletons together would... and time mentors spend with Singletons in their bins. We also sought expertise from many with experience...media. Joe is a lapsed marathon runner, father of twins (AND a singleton), and is proudly "too weird to ...

...frequency of events, instead of relying on clustered dots. In a contour plot, each line contains the same number...machine reads/interprets the signals, the axis is instead divided into positive and negative signal at a ... a ‘gate’; it tells the machine that everything inside the gate is one population and everything outside...which measure light that “scatters” past the cell, instead of fluorescent markers. There are various types...to fluorescent markers and/or cellular dyes and stains. These will be used in a series of one- or two-...different populations using conjugated antibodies against CD11c and CD123. You can see that mDCs are CD11c...

...alkyne-phenol, to label proteins with an alkyne group for click chemistry reactions instead of biotin — a valuable...locations. C) Enrichment of dual-labeled proteins identifies proteins that had been trafficked from the “source...dual-enrichment and mass spectrometry workflow identifies proteins bearing both labels (which had therefore been ... light, catalyzing the biotinylation of nearby proteins. B) TransitID uses sequential labeling by TurboID...2023 bioRxiv (B-C). They were able to label proteins moving to and from the nucleus, ER, mitochondria...

...approach, which focuses on validating antibodies against markers of the human blood leukocyte populations...cases, we would recommend a knockdown approach instead. In this approach, you’re looking for an antibody...permanent knockdown, consider using shRNA vectors instead. With both siRNA and shRNA, it can be difficult...induce/suppress the expression of many related proteins, not just your protein of interest, which can ...interest, which can be difficult to find for many proteins. In practice, it was necessary for us to combine...primary blood leukocytes. The file shared also contains information about the labeling pattern of several...on human blood leukocytes, including antibodies against these markers. The 371 CD markers characterized...

...Kinases: they regulate many proteins, with ~1/3 of human proteins predicted to be phosphorylated on at...swapping of fluorescent proteins and multiplexing is simple when fluorescent proteins with limited spectral...FRET reporters are comprised of two fluorescent proteins with very specific spectral properties that limit... and the orientation of the donor and acceptor proteins, which can be difficult to predict. Slower Dephosphorylation...

...the ability to generate full libraries of mutant proteins. Researchers from the Whitehead lab developed ... Gibson Assembly: An easy-to-use technique that joins homologous DNA fragments in a single-tube reaction...using Gibson assembly is that the longer the oligo insert used, the more likely it will contain random synthesis... enzymes leads to a buildup of uracil and the insertion of uracil into newly synthesized DNA. This labeled...synthesized DNA strand. Each degenerate primer set contains three consecutive randomized bases (NNN) at a ...that the BbvCI enzymes nick the DNA strand that contains their respective recognition site. This means ... matter? Most of the time no, but there are 2 instances where it does. 1) When the backbone vector has...

...are only expressed when the targeting vector is inserted downstream of a functional promoter (gene traps...2008). While the biology behind this AAV effect remains unclear, it is likely not a coincidence that the...gene. pAAV-SEPT-Acceptor, the preferred vector, contains a promoterless splice acceptor-IRES-neoR gene,... CRISPR-based approaches for the generation of knockins via homologous recombination with a homologous...application - the creation of sequence-specific knockins - for which the AAV approach is most well suited... CRISPR-based approaches for the generation of knockins, it is not always ideal. Because of these limitations...devised methods to deliver CRISPR components as ribonucleoproteins (RNPs) that can be supplied separately from...

...reaction that cleaves a nearby RNA sequence that contains a fluorophore that is quenched when intact and...improves sensitivity of the assay. This reaction also contains SSB, strand displacement DNA polymerase, recombinase...fluorescence visualization. In this technique, however, instead of using lateral flow strips to detect the cleavage...Reverse-transcribed RNA samples are amplified by PCR instead of RPA. Unlike RPA, this technique uses Taq polymerase... which is widely available and less expensive. Instead of using the typical PCR machines, the authors ...depend on collateral cleavage by the enzyme and instead relies on its activity on the target regions thus...COVID-19 using CRISPR diagnostics. https://www.broadinstitute.org/files/publications/special/COVID-19%20detection...

...where they bind to their targets, most commonly proteins. You can then use fluorescent microscopy to look...visualize their target. These antibodies are generated against a specific antigen and then conjugated with a fluorescent...but is not attached to a fluorescent molecule, instead a secondary antibody that targets the primary is...for each primary antibody could get out of hand. Instead, it is more efficient to generate a handful of ... multiplexing is that you can ask how multiple proteins relate to one another in the same sample. For ...see Im, et al. 2019.  Fixation Good fixation maintains sample morphology with minimal impact to the target...spectral properties match your available imaging instruments and the spectral properties do not overlap with...

...MTRIA for short), where a fluorescent protein is inserted into one of the loop regions of any G-protein-... Figure 1: MTRIA biosensors are generated by inserting a circularly-permuted GFP into an intracellular...calcium reporter Near-infrared (NIR) fluorescent proteins promise better performance for in vivo use and...indicator using small monomeric NIR fluorescent proteins (miRFP670nano and miRFP720) and the Ca2+-sensing...structure-guided engineering of potassium binding proteins, they developed a family of fluorescent potassium...et al. 2022). By fusing two bright fluorescent proteins with different thermal quenching rates (mNeonGreen...Additional resources on the Addgene blog Fluorescent Proteins 101: Fluorescent Biosensors Resources on Addgene.org...

...plasmid, into which the GOI is inserted, and a Ti "helper" plasmid, which contains Vir genes necessary for T-DNA...gRNA scaffold sequence and allow researchers to insert targeting oligos with minimal cloning required....Dicot Genome Editing Kit from the Stuttmann lab contains a variety of Agrobacterium-compatible, Cas9 containing... T-DNA processing and insertion into the plant genome. B) The Ti plasmid and helper are transformed into...the Vir genes on the helper plasmid and used to insert the intervening region into the plant genome. Created...- promoters used, precise protein sequences or domains, and methods of delivery - the underlying technique...

...studies or make a reporter construct. Fluorescent proteins (FPs) are also the foundation of multiple important...biological research. Photoconvertible fluorescent proteins Newly developed photoconvertible FPs like PsMOrange...repeat the process with these new photoconvertible proteins. With Sattarzadeh et al.’s discovery that GFP ...After exciting GFP-labeled plant cells at 405 nm instead of 488 nm, Sattarzadeh et al. noticed that GFP ...encoding one of the unexpectedly photoconvertible GFP proteins to make a new transgenic line could avoid problems...as well as a wide variety of other fluorescent proteins for your imaging needs. If you need an FP refresher...

...Llamas Required - Synthetic Nanobodies Against Membrane Proteins More resources from addgene.org  Plasmids...single-domain antibodies genetically fused to fluorescent proteins and used to visualize target antigens in living... transgenic mice that express camelid variable domains (aka nanomice), which gives researchers the “best...made to generate the nanomouse. (VHH = variable domains of heavy chain only antibodies) (B) Nanomice express...widely adopted. So thank you to the camelids who inspired this versatile family of tools, the applications...

...and to disrupt endogenous genes at the site of insertion. In both of these cases, transposons combine the...blue) in a plasmid backbone. A separate plasmid contains the transposase gene (red) for expressing the ...sequence is duplicated near the gene of interest insertion site.   The Sleeping Beauty transposon system ...within the genome. The Sleeping Beauty transposase gains its name from the fact that it was recreated from...continues to be altered in order to improve its gene insertion efficiency in primary cells. In 2009, Zsuzsanna...or 5’ end of the gene of interest allowing for insertion in the correct orientation. These plasmids use...

...Carolina (plasmids at Addgene). It consists of two domains, LOV2 and Zdk1, where Zdk1 binds LOV2 with high...normally sits on microtubules to attract other proteins involved in their growth and extension. Now, they...domain. Each half was fused with one of the LOVTRAP domains. When both halves of the modified EB1 protein are...inactivated by the separation of its synergistic domains. This paper demonstrated that the migration of ...fluorophore binding aptamers and MS2-binding fluorescent proteins were the state of the art in RNA imaging in vivo...on the lookout for better, brighter fluorescent proteins (FP) with improved tissue penetrance and live ...

...Argonaute proteins are also present in prokaryotes, and some of these proteins protect against invading...reasons why you might choose NgAgo over CRISPR proteins Cas9 or Cpf1 - keep reading to learn more! Background...: Argonaute and NgAgo In eukaryotes, Argonaute proteins play an important role in RNA interference, binding...when the protein is being synthesized, so NgAgo remains faithful to its guide DNA and cannot switch guides...

...usage has been limited by technical problems. For instance, it’s much more difficult to introduce a gene ...strategy for transducing primary cells. What if, instead of supplying a DNA construct, they could simply... adapted from D’Astolfo et al. In certain instances, the potential benefits of nonviral transduction...with virus work. Fourth, working directly with proteins removes the limitations of packaging size associated...References Efficient Intracellular Delivery of Native Proteins. D'Astolfo DS, Pagliero RJ, Pras A, Karthaus WR...PubMed. Find the Plasmids @Addgene: iTOP-tested proteins and reporters Previous Protein Delivery Techniques...

...Tom, and Alan E. Tomkinson. "Eukaryotic DNA ligases: structural and functional insights." Annu. Rev. Biochem...many molecular biology protocols, including vector-insert joining for recombinant plasmid construction (restriction...DNA ligase, are naturally able to discriminate against ligating substrates containing base pair mismatches... can be achieved through a combination of the intrinsic selectivity of the ligase active site and careful...indicated that the optimal buffer for Taq DNA Ligase contains 100 – 200 mM KCl at pH 8.5. This optimization....6 (2004): 757-764. PubMed PMID: 15582400. 2. Tomkinson, Alan E., et al. "DNA ligases: structure, reaction...