...BioRender.com. Transformation Grade DNA (low quality/low quantity) – Bacterial transformation protocols require...eluting the DNA from the substrate. In a column-based approach, the substrate is a silica matrix fixed... large amounts of high quality DNA. Figure 1 illustrates the three grades of DNA you may need based on...highest-competency cells you will still struggle to get transformants if the plasmid you are using is completely degraded...important reactions like PCR or restriction digests. Transfection Grade DNA (high quality/high quantity) – Depending...Depending on the experiment, mammalian cell transfections can require quite large amounts of DNA (easily...the plasmid DNA from your cleared lysate to a substrate, washing away left-over debris that carried over...

...signal downstream of the transcription start site will terminate transcription of most of the ncRNA (Figure... schematic illustration of CRISPR/Cas9-based approaches to study ncRNAs. (A) Transcription start site ... coming, right? Non-coding RNAs (ncRNAs) are transcribed but do not encode proteins. They play important...Using a single guide RNA (sgRNA) to create a double-strand break works well because very small deletions and...sequence can completely disrupt expression of the transcribed ncRNA (Jiang et al. 2014).  Simple knock-out ...lncRNA regulatory elements that are crucial for transcription and downstream function. Rather than attempting...the lncRNA locus, scientists can mutate the transcription start site, splice sites, exons, and/or promoters...

... a strong transcriptional activation domain. Guided by dCas9, VP64 recruits transcriptional machinery ...as two other potent transcriptional activators (p65 and Rta). These transcriptional activators work in ...interest. Upon binding, CRISPRa systems recruit transcription factors to increase gene expression. CRISPRa...CRISPRa methods vary in their transcriptional activators: some methods rely on fusion proteins while others...gene regulation. This can be used to activate transcription during either initiation or elongation, depending...uses specially engineered sgRNAs to increase transcription. This is done through creating a dCas9/VP64 ...at each loci of interest, this allows more transcriptional machinery to be recruited per targeted gene...

...18930153. PubMed Central PMCID: PMC2644460. 12. Querido, Emmanuelle, and Pascal Chartrand. "Using fluorescent...folded beta-barrel structure, followed by an intramolecular reaction that generates the chromophore (Tsien...and require uptake by the cells. Which of these strategies works best for a specific biological system under...intensity of purified protein at different acid concentrations. Overviews of the pKa values of different fluorescent... tag and several others. Another peptide-based strategy uses an enzyme to covalently label the peptide...specific RNA stem loop-structure (Querido and Chartrand, 2008). Finally, a nuclease-deficient Cas9 can... Since longer wavelengths have better tissue penetration, (infra)red fluorescent proteins are also a better...

...is composed of single stranded DNA, which is particularly well suited for strand invasion, a key step ... are then packaged into AAV virions via transient transfection of 293T cells with helpers, and used to...identical targeting vectors delivered through transfection of naked DNA. Other investigators who had been...inserted downstream of a functional promoter (gene traps). The resultant gene targeting system could be used...the efficiency of homologous integration via gene trap enrichment, whereas pAAV-TK-Acceptor has a conventional...specific frameshift mutations through aberrant double strand break repair, it has proven more challenging to...AAV-based gene editing via the introduction of a double strand break by using a targeted nuclease. Asuri et al...

...found in the extracellular space. The targeted epitopes can be motifs within transmembrane proteins, such...nature, antibodies cannot penetrate the cell membrane; hence, intracellular labeling with antibodies requires...label intranuclear proteins of interest, you can label cytosolic proteins alongside the intranuclear ones...such as receptors, or post-translational modifications on those proteins, like glycosylation patterns....cells. However, there are more techniques and strategies one can employ beyond simply incubating cells...downstream applications, like an in vivo adoptive transfer of those CD8+ T cells, require the cells to be...your sorted cell sample than MACS. An effective strategy to sort out your cells of interest via FACS using...

...Modular fluorescence unit fused with TRansmembrane region-to-IntrAcellular loop linkers” (MTRIA for short),...-gTEMP plasmids here!   Lu K, et al. Intracellular Heat Transfer and Thermal Property Revealed by Kilohertz...new class of sensors for neuropeptides and neurotransmitters. Motivated to develop a biosensor for oxytocin...sensors based on GPCRs recognizing various neurotransmitters, neuropeptides and other ligands - almost ...inserting a circularly-permuted GFP into an intracellular loop of various G-protein-coupled receptors....fluorescent sensor for real-time measurement of extracellular oxytocin dynamics in the brain. Nat Methods....kinetics and localization for imaging synaptic transmission. bioRxiv 2022.02.13.480251. doi: 10.1101/2022.02.13.480251...

...genes from a single transcript, or if you want to prevent read-thru transcription from resulting in the...developing a general translation initiation sequence known as the Species-Independent Translational Sequence (SITS...extensive sequences used for cloning into the final translated products. Negative selection markers built into...3509-3514. Tikhonova, Elena B., et al. "A transcription blocker isolated from a designed repeat protein...of which you can clone your gene of interest. Translation of the standardized leader peptide prevents secondary...encoding your gene of interest from affecting its translation initiation and instead makes initiation heavily...the production of a downstream peptide, the transcription terminators in this series are just the tool...

...techniques used to generate transgenic mouse lines, such as BAC transgenic or traditional knockins/knockouts, ...researchers generating transgenic animals may choose Southern blotting to screen founder transgenic animals for ...fragmented into smaller pieces, run on a gel, and transferred to a membrane for analysis through hybridization...for the inserted transgene because Southern blots have a lower false positive rate than real-time PCR (4...hydroxyl group required for the extension of a DNA strand (8). Adding a small amount of a single radiolabelled...regular nucleotides results in the synthesis of DNA strands of all possible lengths because extension is terminated...prevails. Sequencing whole genomes or profiling transcriptomes (RNAseq) require sequencing of many samples...

...’s lab at Temple University has demonstrated two potential strategies for CRISPR-HIV therapeutics - one...one using dCas9-SAM to activate HIV-1 transcription and destroy infected cells, the other using wild-type... no viral protein. To solve this problem, two strategies have been proposed. The first, “shock and kill...cytotoxicity or the immune response. The second strategy is the simple removal of the HIV-1 genome from...histone deacetylase (HDAC) inhibitors to increase transcription of the integrated HIV-1 genome. For a targeted...dCas9-VP64 fusion commonly used to activate transcription, but with two additional activation domains ...domains (MS2 and p65) to enhance transcriptional activation. Zhang et al. designed multiple gRNAs to target ...

...proteins, which are directly trafficked to cell membranes. Membrane trafficking of farnesylated derivatives... virus post-transcriptional regulatory element (WPRE), commonly used to increase transgene protein levels...has become very useful in lineage tracing; “Zebrabow” was used to trace the development of the corneal epithelium...Brainbow 3.0, 3.1, 3.2 plasmids References: Transgenic strategies for combinatorial expression of fluorescent...2013 May 5. PubMed. Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish... also been applied to cell marking and lineage tracing in other systems, such as zebrafish and Drosophila...This system differs from Brainbow-1.0 in that transient Cre recombination is required to prevent constant...

...different DNA strand comes along, it can attack this covalent bond thus joining the two DNA strands and releasing...planning to transform right away OR you can store the reaction at -20C overnight. 4. Transform TOPO Cloning...end cloning. So how does TOPO cloning work? As illustrated in the Figure below, the “A” overhang on the ...DNA sequence 5´-(C/T)CCTT-3' and digests double stranded DNA at this sequence. The energy from this breakage...stored as a covalent bond between the cleaved 3′ DNA strand and a tyrosyl residue of topoisomerase I (1). If...researcher to perform blue-white screening after transformation - self joining of the vector ends results in... minutes will not significantly improve the transformation efficiency).  5. Select and Analyze 10 White...

...of DNA, transcription of DNA to make RNA, translation of RNA to make proteins. That's central to everything...Science describing the development of “transcriptors,” transistor-like digital genetic switches that enable...comprised of well-characterized bacterial transcription and translation initiation elements. Open science with.... We have a strategy that organizes how we start the work, and it begins with the central dogma - replication...reliable gene expression via standard transcription and translation initiation." Nature Methods. 10, 354...cellular computing. These logic gates built from transcriptors are now available at Addgene through the Biobrick... handle on precise, forward engineering of the central dogma at the genome scale, it's going to be tricky...

...contains 11 β-strands and one central ɑ-helix that can be split into 3 parts: (1) β-strands 1-9 (β1-9) and... and the one ɑ-helix, (2) β-strand 10 (β10), and (3) β-strand 11 (β11). Each part contains components ...developed, such as the Forster resonance energy transfer (FRET)-based executioner caspase reporters. In...GFP (GFP1-9 and GFP10-11) by flipping a single β-strand of GFP, hence the name FlipGFP. FlipGFP increases...from β1-9 they will rapidly recombine with β1-9 strands nearby resulting in fluorescence within tens of...11 are antiparallel. To prevent binding, the β strands in FlipGFP were redesigned so that they are parallel...E5 and K5) that dimerize together and force the strands into a parallel conformation. Between β11 and one...

...containing transcription factor binding sites can increase transgene expression and transduction efficiency...as the gene that they package. In a transfer plasmid, the transgene is flanked by two ITRs, and the ITRs...modifications can drastically affect gene transfer efficiency and transgene expression (Zhou et al. 2017). For...these mutations can affect transduction efficiency and subsequent transgene expression. Replacing the ...nucleotide sequences that flank the ends of the single stranded DNA genome of AAV in the native virus. In an AAV...efficiency (Shitik et al. 2023). Transgene expression can be altered by using ITR sequences from different AAV...Most ITR sequences are from AAV2 because they demonstrate high promoter activity (Earley et al. 2020), ...

... of the four strands of the cleaved duplex, while the fourth strand (the released strand in Figure 1 below...discovered that single stranded donor DNA: Complementary to the released strand, 127bp in length, With...single or double stranded donor DNA with other design parameters. Example single stranded donor DNA and ...biological process in detail often suggests new strategies to manipulate that process. We began our research...investigations into the release of DNA post-cut. Using substrate DNA labeled on both sides of the break, we determined...presented in Figure 2. Figure 2: Single-stranded donor DNA interaction with the Cas9-DNA duplex...replacement without cutting Our in vitro results also demonstrated that catalytically inactive dCas9 produced a...

...of scientist training in the US and around the world. There are rumors that we are training too many scientists...few of these trainees are sufficiently prepared during their 6-12+ (!) years of training to get jobs. ...developing transferable skills to enable pursuit of these diverse opportunities after training. One of the... examples of science trainees taking a proactive role in their non-bench training. Grad students at Washington...academic administrators who are finding ways to provide new types of career preparedness training for their...the way we view the graduate school and postdoc training years. There is no doubt that there are too many...academia. Certainly funding for academic research and training is getting tighter and competition is fierce. ...

...constructs to visualize cell cycle dynamics, titrate inducible transgene expression, or a variety of other clever...throughput cloning strategies There are several high-throughput cloning strategies available for assembling...PubMed PMID: 25909630. PubMed Central PMCID: PMC4409215. 7. Swartz MN, Trautner TA, Kornberg A. Enzymatic ...incorporate each element into the final construct. This strategy can be used to build vectors with diverse functions...containing a unique fluorescent protein with minimal spectral overlap between them (Table 1). In each construct...expression) and a polyA signal (to terminate transcription) and the four constructs were then combined ...suited for particular downstream applications. The tradeoffs between various cloning methods are listed in ...

...detect a variety of intracellular conditions, including ion (such as Ca2+) concentrations and pH, using a ...Developmental/transgenic uses: Because of its stability, GFP can be used in lineage tracking capacities ...reporter for DNA double strand break repair, and to label pathogenic intracellular microbes to visualize...spontaneously and without additional co-factors, substrates, or enzymatic activity – it only requires the...folding at 37C, reduces aggregation at high concentrations T203Y Red-shifts the wavelength (yellow derivatives...K26R, Q80R, N146H, H231L, (and probably others) Neutral mutations    A multitude of applications Due...backbones for constructing fluorescent fusions. Transcription Reporter: Placing GFP under the control of a...

...control than is achieved by traditional pharmacologic methods. A number of strategies have been developed for... in the extracellular domain which activates the native signaling cascade of the intracellular part of...and Ca++ to passively enter the cell with the concentration gradient to depolarize it (Nagel et al., 2003...pumps that pass ions (Cl-, H+, Na+) against the concentration gradient and hyperpolarize the cell (Mattis ...activation by light showing ions flowing with the concentration gradient, and of activation inhibitory (ArCH...ArCH) proton pump passing ions against the concentration gradient. Created with BioRender.com.   Opto-G-protein...GPCR) regulate cellular function through multiple intra-cellular signaling cascades. Optical methods for...