...BioTechniques 2015; 58:161-170. PubMed PMID: 25861928. 3. Jiang W, Zhao X, Gabrieli T, Lou C, Ebenstein Y, ...neighboring fragments can be created via PCR amplification with primers that contain the appropriate homologous... tools that help you design primers for PCR amplification of fragments to incorporate such regions of ...assembly meets CRISPR Gibson can be adapted to more complicated cloning schemes, such as those where the vector...CRISPR technology and is described in a recent publication by the Lockey lab (Wang, et al. 2015). In this...aspects of the SLIC methods. First, it uses a dedicated 5’ exonuclease instead of using the exonuclease...DNA fragments contain these sites at the correct location and NOT in the middle of your insert. Gateway ...

...without the risks associated with prolonged expression. 3. Stable expression in non-dividing cells IDLVs can...decades. Widely used across various research applications, they have become indispensable tools for manipulating... easily, without the need for time-consuming purification processes, and are powerful, due to their ability...-lasting changes in cells. However, for some applications, integration might be considered a double-edged...dividing cells because they lack an origin of replication (Banasik and McCray, 2010). However, IDLV genomes... certain types of clinical or basic research applications. Considerations for IDLV use If you’re reading...Integrase-defective lentiviral vectors: progress and applications. Gene Therapy, 17(2), 150–157. https://doi.org...

...base editor development going forward. Figure 3: Mechanism of gGBE. Created with BioRender.com. ...Red arrows indicate a conversion directly catalyzed by the base editor. Black arrows indicate natural cellular...researchers can now select the best tool for their application from among many options.  Conclusion With these...engineered human uracil DNA glycosylase. Nature Communications, 15(1), 4897. https://doi.org/10.1038/s41467...via engineered uracil-DNA glycosylase. Nature Communications, 15(1), 6397. https://doi.org/10.1038/s41467...

...Model for Regeneration and Aging. Gerontology, 66(3), 212–222. https://doi.org/10.1159/000504294 Wang,...studying genetic modification for their ease of manipulation and their ability to be replicated. Because E...., 2004). Additionally, breakthroughs in gene modification tools and methods such as CRISPR have vastly...Escherichia coli as a Model Organism and Its Application in Biotechnology. In Escherichia coli - Recent...Physiology, Pathogenesis and Biotechnological Applications. InTech. https://doi.org/10.5772/67306 Koornneef... bacteria with superior performance. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-04899...

...protein." (1998): 509-544. PubMed PMID: 9759496.  3. Buckley, Anthony M., et al. "LOV-based reporters ... proteins.Therefore, GFP is not suitable for applications that require anaerobic conditions. In experiments...an additional protein module that can report on location. This approach enables the study of proteins in...fusion with GFP allows for detection of genomic locations (Chen et al., 2013). GFP fluorescence overlaps...case when proteins undergo post-translational modification with fatty acyl chains. For instance, myristoylation...C-terminal consensus sequence (-CaaX). Both modifications are disrupted when GFP is fused to these sequences...

...strain. F- Δ(ara-leu)7697 [araD139]B/r Δ(codB-lacI)3 galK16 galE15 λ- e14- mcrA0 relA1 rpsL150(strR) spoT1...that carried out the labor-intensive process of replicating your new plasmid: the Escherichia coli bacteria...conjugation. Genes listed on F´ are wild-type unless indicated otherwise fhuA (formerly tonA) ferric hydroxamate...vectors. Used by Addgene for pooled library amplification. F' mcrA Δ(mcrBC-hsdRMS-mrr) recA1 endA1 gyrA96...typically standard; just having the gene listed indicates it is non-functional. If a gene is deleted, it... Most Popular Plasmids 101 Posts: Origin of Replication The Promoter Region – Let's Go! Mammalian Vectors...

...separated by size, (2) transferred to a membrane, and  (3) detected using antibodies.   Figure 1: Overview...post-translational modifications, quantify protein levels relative to standards, detect the cellular location of proteins...pull-down assays.  Westerns are divided into two categories, native and denaturing. In a native western, ...matrix, the blotting membrane, the gel, a copper cathode/iblot gel matrix, and filter paper. Pretty much...search, first pay attention to the validated applications listed on the vendor’s website and choose an...western and vice versa.  In addition to validated applications, pay close attention to the controls listed ...and incubation times. If the vendor includes publications that cited the antibody, consider reviewing ...

...scientific enterprise and not just impact. Cause #3: Poor experimental design It’s easy to get excited...someone has replicated it precisely. You can find a great discussion of different types of replication in Nosek...perfect story for a “high impact” publication can lead to the publication of rare flukes showing positive...that fall into this category include performing experiments with too few replicates leading to suspect ...these positive results will be published. The publication record will then show that the tested hypothesis...reproducible publishing practices. One example is the publication of peer reviews so we can see how and why papers... also means that you’re more likely to get a publication (for better or worse, the currency for success...

...Originally published May 3, 2017 and last updated Sep 24, 2020 This post was contributed by guest blogger...the optimal location. It is also important to have good information on the exact location of the TSS. ...appreciable levels of modification. Whole-genome sequencing of cells modified by CRISPR indicates that the consequences...specific base edit, or modulation of gene expression. Location and sequence are important considerations for ... gRNAs. For indels, it's not so important what location in the gene you target, but it is important that...fewer sequences to choose from). Finally, for HDR, location is much more important because you have to target...target site is far more constrained by the desired location of edit; efficiency decreases dramatically when...

...spend the next 6 years. Know yourself I rotated in 3 labs before choosing a graduate school lab. All the...honestly evaluate your own style of work and communication before making this important decision. I’ll ...Prepare by going sleuthing on the lab website, publication records, LinkedIn, ResearchGate or Twitter. Be...the appropriate first authorship position on a publication of her project and made another lab member first...– including failure to acknowledge good work, allocation of meaningless tasks, removal of responsibility...connections such as serving on Boards? Does the publication record show evidence of industry collaboration...skills guide Learn about careers in science communication Read posts on mentoring for scientists Resources...

...image-based figures for scientific publications. PLOS Biology, 19(3), e3001161. https://doi.org/10.1371...protein is (localization) and how much there is (quantification). But what if you’ve got several interesting...primary antibodies that have been validated in IF applications (find IHC validated or ICC validated antibodies...Immunocytochemistry Protocols for other antibody applications...

...and can exhibit recombination levels as high as 1-3%. In light of this finding, we now include guidance...promoter, such as the truncated tyrosine hydroxylase (TH) promoter for catecholamine neurons or glial fibrillary... Flp), to either flip or excise a gene that is located between them. Early recombinase-dependent vector...expression profile at multiple steps from plasmid amplification in bacteria to transgene expression in vivo.... the level of spontaneous recombination. This indicates that the amount of leaky expression can largely...are typically employed during viral plasmid amplification (Fischer, KB. 2019). This means that all DIO...of spontaneous reversion during AAV plasmid amplification. On the left homologous pairs of scrambled cre-recombinase...

...about roles in this field, most jobs needed at least 3 months experience working in a developing tropical...at the Institute of Cancer Research in Sutton, located on the edge of London to gain relevant experience...recession. As each advertised job received many applications, employers were requesting that only scientists...commercial company. In my particular situation, communication between departments was sometimes difficult ... order to have meetings between teams. This communication is vital because otherwise I’d lose touch with...

...Science 6.1 (2015): 50-69. Royal Society of Chemistry. 3. Ermolenko, Dmitri N., et al. "Observation of intersubunit...cellular impediments. FRET has been used in many applications, including drug discovery by big pharma (i.e... acceptor doesn’t need to go to the same exact location as the donor. As long as some of the acceptor ...C-terminus of your target protein, but even these modifications can affect the structure or function of certain... close to the donor, faulty delivery to other locations in the cell are not a problem, as long as they...The poly-histidine tag often used for protein purification is a reliable FRET acceptor labeling site when... way as NTA-sepharose beads used for protein purification: the NTA binds to the His tag on your protein...

... dynamics in mammalian cells. Biophysics Reports, 3(4–6), 64–72. https://doi.org/10.1007/s41048-017-0037...emission wavelengths, which are pivotal in their application in biotechnology (Grimm & Lavis, 2022). These... substrates/peptides and antibodies. Example applications of this include coupling to an antibody, whereby...whereby excitation of blue light will indicate where FITC is bound through green light emission. Key considerations...typically bind to. Stability of fluorescence: For applications that involve ranges of pH, and significant lengths...light, is another factor to consider. If your application requires longer periods of exposure, you’ll want...degrees of β-arrestin-2 recruitment to receptors, indicating their agonistic nature. The potency and efficacy...

...should be ~60°C (between 50 and 65, with no more than 3 degrees between the two Tms)  Aim for 50-60% GC content...single peak, indicating one amplicon and no primer dimers. (b) A shoulder peak indicating primer dimers...Method Standard Curve?  Amplification Efficacy Calculation Amplification Efficacy Assumptions ...understanding and technical proficiency with common applications - now let’s dive right in! You know qPCR is...is most commonly used to measure relative quantification of mRNA, which can be used as a proxy measure...as it solves, so understanding and using the application correctly is imperative. Picking your chemistry... at least 25 bp away from each primer, ideally located close to either the forward or the reverse primer...

... expanded version by Nyla Naim was published Sept 3, 2020.   Scientists around the world have been making...light has limited applications in vivo. UV and blue light is readily absorbed and scattered by the skin, so...strategies Publication System nickname Photoactivatable moiety Cas9 variatn CRISPR protein application Split...improvements to CRISPR technology since its initial applications for genome engineering in 2012.  Many of these...scientists to use light to externally control the location, timing, and reversibility of the genome editing...enzymatic activity. Photoactivatable genome modifications by NHEJ and HDR using split systems Later in...mutations via NHEJ (frequency of 20.5%) and induced modifications by HDR (frequency of 7.2%). The authors additionally...

...or 1kb fragments are produced (see figure above). 3 Reasons The PSU Ladders Are Awesome Tools: They are... plasmid or group of plasmids' functions and applications. We hope that these articles make it easier ...FLIM (Fluorescence Lifetime Imaging Microscopy) applications. To demonstrate the power of mCyFP1, the Lin... Coselection efficiency is so high that some applications may permit working with edited “pools” of cells...showed high editing efficiencies with AsCpf1, indicating that ouabain coselection is a robust and flexible...be found at Addgene and in Figure S12 of the publication. Agudelo D, et al. Nature Methods. 2017. PubMed...potential of base editing for precise genome modification. Plasmid ID Plasmid Name Cas9 Variant Speacial...