...Dawson lab Re-transformation of the SPLINTR Libraries Protocol.   Selecting your strain for amplification...ensure the highest possible transformation efficiency. Using Stbl4 or other ultra-high efficiency cells ensures...libraries, chemical transformations can also be used. Efficiency for chemical transformations is less than that...for your transformation(s). The amount of competent cells you need total and per transformation may vary...the manufacturer’s protocol for transformation and expected transformation efficiency, or more specifically...with a large backbone might be more difficult to transform.    Figure 1: Factors to consider when ... coverage, since this will help decide what transformation efficiency you need to achieve. Most pooled...

...Chemical Genomics and Translational Medicine 3.1 (2009). PubMed PMID: 20161833. PubMed Central PMCID: PMC2802762...23667515. PubMed Central PMCID: PMC3647052. 11. Stryer, Lubert. "Fluorescence energy transfer as a spectroscopic...is it? What parts of it move when you feed it substrate or add an essential cofactor? How many binding...what's it good for? Fluorescence resonance energy transfer (FRET) is a biophysical interaction between two...first fluorophore (the donor) is excited, it can transfer energy directly via resonance to the second fluorophore...LanthaScreen) (Degorce et al., 2009), measuring intracellular calcium (Miyawaki, Griesbeck, Heim, & Tsien,...structural changes in the ribosome during protein translation (Ermolenko et al., 2007). All that is required...

...genetically tractable. Mice can be easily manipulated with tools like CRISPR to make transgenic lines. The...28804628. PubMed Central PMCID: PMC5525645. Mello, Craig C., et al. "Efficient gene transfer in C. elegans...elegans: extrachromosomal maintenance and integration of transforming sequences." The EMBO journal 10.12 (1991...externally. Fruit flies are also relatively straightforward to manipulate genetically. Scientists can make... in only 6 weeks compared to months to make a transgenic mouse line. Over the years the fly has become...DNA or RNA to edit their genomes or to create transgenic animals. Zebrafish are an ideal model organisms...development studies as their embryos are completely transparent. Thus scientists can easily observe early developmental...

...to measure stickiness. In vitro, ultracentrifugation or gel filtration is the most common analytical method...random insertions can be generated with a transposon-based strategy (Sheridan, 2002). Avoiding protein over-expression...polarization independent of GDI-mediated extraction and actin-based trafficking." PLoS biology 13.4 (2015): e1002097...physiologic conditions." Traffic 13.5 (2012): 643-649. PubMed PMID: 22289035. PubMed Central PMCID: PMC3324619...vitro transposition reaction." BMC neuroscience 3.1 (2002): 7. PubMed PMID: 12086589. PubMed Central PMCID...engineering of fusion proteins is relatively straightforward. However, generating a fusion while keeping...interest can be challenging. In this blog I discuss strategies to generate fusion proteins and highlight some...

...MSN2 transactivation domain. Transcription factor activation via the appropriate ligand - estradiol for...GAL4/estradiol controlled transcription factor and a ZIF268/progesterone controlled transcription factor...ZIF268. To demonstrate independent control of gene expression, Aranda-Díaz et al transformed yeast with...specific to each transcription factor. They then induced cultures of these transformants with various concentrations... levels. That is, if transcription factor 1 controls expression of transcription factor 2, which directly...pH-based biosensors used to detect the maturation of neutral vesicles to more acidic lysosomes. Though a number...Tyler J. Ford Listen to the Synthetic Yeast Transcription Factors Podcast Segment Researchers from the...

...between the edited strand and wild-type strand of the host DNA. When these strands hybridize, the nuclease...PubMed Central PMCID: PMC4349094. 3. Bell, Charles C., et al. "A high-throughput screening strategy for ... known as CRISPR genotyping, is critical to demonstrating causal relationships between genotype and assayed...nuclease can detect strands with mismatches and cleave them. The results are then visualized using gel electrophoresis...is also key to perform these validations as translational researchers begin to use CRISPR as a therapy...nucleotides) that may still lead to off-target double-stranded breaks (Tsai et al. 2015). Additionally, current...et al. 2015) for cell-based experiments. These strategies can be used in concert with in silico prediction...

...showed that the DNA extract is a heterogeneous mix of single-stranded and double-stranded species. While not...molecules in the DNA extract associate with complementary strands, enough double-stranded species exist in...previously require a preliminary double stranding step. The double stranding step was assumed to be necessary...genome is single-stranded and both TA-based ligation and tagmentation require a double-stranded template.  ... [-] strands that are packaged at the same frequency, we reasoned that, following DNA extraction, the ...the [+] and [-] strands might naturally associate forming a double-stranded species that could be used as... of the main tenets of our quality control (QC) mantra is to do our utmost to ensure that scientists are...

...humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator)-containing...Multiplexing from a single transcript You can also multiplex gRNAs via a polycistronic transcript. Rather than being...being transcribed from different promoters, the gRNAs are transcribed together and are flanked by specific...simultaneously. Vectors for both transient expression and Agrobacterium-mediated transformation are available. For ...system for processing a continuous multi-gRNA transcript, each gRNA must be expressed from its own promoter.... But that doesn’t mean you have to clone and transfect multiple promoter-gRNA constructs in order to ...destination vector that expresses Cas9. This is illustrated in the schematic below.   Figure 2: ...

... Infectious titer: the concentration of viral particles that can transduce cells. Infectious titers ...PMID: 22428975. PubMed Central PMCID: PMC3640491. 2. Grimm, D., et al. "Titration of AAV-2 particles via...PubMed Central PMCID: PMC5445070. 6. Wang, Feng, et al. "A reliable and feasible qPCR strategy for titrating....qPCR...Digital droplet PCR...DNA dot blot...Transduction assay...SDS-PAGE...Electron microscopy… Any ...can AAV titers tell you? Physical titer: the concentration of viral particles containing viral genomes....Physical titers are measured by quantifying the concentration of viral genomes (by qPCR or other DNA quantification...titer of a viral vector stock can vary based on transduction target and can also be altered by freeze-thaw...

...dimerization.   1. Transmembrane fluorescent protein fusions Transmembrane proteins, i.e. receptors...receptors and transporters, are integrated into lipid bilayers. Fusion of an FP to a transmembrane protein significantly...physiologic conditions. Traffic, 2012. 13(5): p. 643-9. PubMed PMID: 22289035. PubMed Central PMCID: PMC3324619...Green Fluorescent Protein (GFP/EGFP) for a transcriptional reporter or as a general cytoplasmic label ...too common issues follow. It's a matter of concentration Most FPs in nature are prone to dimerization...dimerization (i.e. EGFP) [1, 2] or even form obligate tetramers (i.e. DsRed) [3]. This is a problem for fusion proteins...termed the dissociation constant or Kd, and its concentration. The smaller the Kd, the more likely molecules...

...poster that Cas9 uses to track down its suspect—the target DNA. tracrRNA: A “trans-activating CRISPR RNA”...target and nontarget DNA strands upstream of the PAM site found in the nontarget strand. Protein Cas: ...sequences and keeping the array organized. Target strand: The strand of foreign DNA that Cas proteins recognize...recognize using an RNA guide. Non-target strand: The paired strand of foreign DNA cut by Cas9, whose sequence...biologists. An sgRNA combines a crRNA and a tracrRNA into a single strand that folds back on itself, simplifying...Regularly Interspaced Short Palindromic Repeats. That contrast of simplicity and complexity is reflected in the... of CRISPR. The bacterial chromosome encodes a tracrRNA (in some systems including Cas9), Cas proteins...

...format of some transcriptional activators: that the DNA binding domain (DBD) and transcriptional activation... haploid yeast fusion plasmid transformants into a diploid strain for screening), or arrayed on a 96 well...flurry of discovery in the field of eukaryotic transcriptional activation and cell biology. During this period... regardless of whether it interacts with a transcriptional activator (1a) or a repressor (1b). Scientists...interactions. In later years, improvements to yeast transformation protocols also helped establish the yeast two-hybrid...classic two hybrid system (also called the interaction trap), genes encoding proteins of interest(POI’s) are... These fusions are then co-expressed in a yeast strain that also contains a reporter gene cloned downstream...

...small, double stranded RNA molecules that degrade target mRNA transcripts. When transiently introduced, ...expression is tunable by titration with different amounts of tetracycline. Tetracycline regulation is reversible...fused to a transcriptional repressor to your gene of interest. It will inhibit transcription instead of...of introducing a DNA double strand break. Transient introduction of CRISPRi or inducible expression (more...proteins. Addition of tetracycline or one of its analogs will induce these transcriptional changes, and expression...baseline) and occurs at the transcriptional level.     Fig. 2 Tetracycline on/off promoters and ...working with an endogenous locus. The most straightforward way to turn off expression permanently is to...

...but also a biological transposon system. Sleeping Beauty is a synthetic transposon system that can be used...reconstructed a transposase system based on the naturally mutated and non-functional transposase gene found...resurrecting this transposon system? Scientists can use Sleeping Beauty transposons as a non-viral system... cause cells and muscles to contract depending on the context. Contraction is critical for processes such...you can use to study these genes! Groucho, a transcriptional co-repressor The first conference room, that...Addgenies, was named after the protein Groucho, a transcriptional co-repressor important in development. Groucho.... It binds to a specific set of DNA-binding transcription factors to repress the expression of genes primarily...

...Determine ultrasensitive and absolute nucleotide concentration of starting material AAV Titrations Allelic...the primers can anneal to the single stranded DNA template strands. DNA polymerase is also stable at this...is a method that provides ultrasensitive and absolute nucleotide concentration, unlike qPCR where results...currently uses ddPCR for AAV titrating. You can learn all about ddPCR for AAV titration and find helpful tips...template DNA to amplify from and unique single stranded DNA primers that bind upstream (5’) and downstream... process as DNA polymerases require an existing strand of DNA to add nucleotides to.  Using these reagents...reaction:  Denaturation- To amplify DNA, the two strands of the template DNA first have to be separated....

..., expressing Ad E1A and E1B, can be transfected with your transfer plasmid, carrying your gene of interest... performed 12–18 hours post-transfection to help remove toxic transfection reagents and keep the cells...infectious titer, meaning the concentration of viral particles that can transduce cells, which can vary based...the AAV Transfer PlasmidAAV Variables That Matter AAV protocols on addgene.org  AAV Titration by qPCR ...TechnologyAAV Titration by ddPCRAAV Purification by Iodixanol Gradient UltracentrifugationAAV Production ...helper plasmids, commonly called “triple plasmid transfection” (Figure 1).    Figure 1: Overview of key steps...production, iodixanol gradient purification, and AAV titration by qPCR.   Pro tip! An optional media exchange...

...in an unmodified bacterial strain would result in read-through translation of many stop codons and detrimental...terminating translation in response to the presence of TAG sequences in ORFs. In this bacterial strain, TAG ...mutagenesis. In the C321.ΔA strain, this TGA will be recognized by Sep-tRNA and translated with a specific phosphoserine...one of the most inclusive databases of post-translational modifications, identifies a staggering ~250,... enabled researchers to create an orthogonal translation system (OTS) for the incorporation of phosphoserine...system defined three components required for translation (Figure 1): A tRNA that recognizes a specific...elongation factor that allows proper extension and translation of the pSer-incorporated polypeptide. These ...

... performed on whole-cell extracts from stable pools of A549 cells transduced with Cas9 expressing lentivirus...generated from A549 cells transduced with lentiCas9-blast. A549 cells were transduced (MOI = 37) and selected...generate DNA duplexes with one strand from the edited cells and one strand from the control cells, which...gene in the BRAF-gRNA transduced cells, and no such edits in the cells transduced by the control GFP-gRNA...generated A549 cells stably expressing Cas9 via transduction with Cas9 lentiviral vector, lentiCas9-Blast... Figure 1: Cas9 expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9 Western blotting...considering that A549 cells are relatively easy to transduce. I ended up with too many Cas9 integrations per...

...writers have been curious about the capability of transforming the genetic code of living organisms and its..., the father of scientific fiction, was also a trained biologist. This is apparent in his work The Island...that Streptococcus pneumoniae bacteria could be transformed from one organism to the other. This is now famed...explained how the four letters of the DNA get translated into the twenty amino acids. Once scientists ...what would happen if these corporations would extrapolate genetic engineering to exploit human beings. ...storyline involves the resurrection of dinosaurs by extracting the DNA from fossilized amber and supplementing... DNA sequences while FokI then induces a double strand break. These breaks can then be repaired by homologous...

...in special strains of E. coli that tolerate the expression of the toxin gene. One such strain is DB3.1,...popular lab strains, such as NEB Stable, JM109, XL1 Blue or XL10 Gold, that are F’. These strains could possibly...out our blog post on Common Lab E. coli strains for more strain info. Additional cloning resources As cloning...available for approaching it. These include the traditional restriction enzyme/ligase-mediated method, the.... One of the most time-consuming aspects of traditional cloning is the identification of clones that ...poison, locking up DNA gyrase with broken double stranded DNA and ultimately causing cell death. ccdA, another..., et al. showing the pKIL 18/19 vectors and demonstrating the concept of restriction enzyme-mediated disruption...