...the reporter mRNA is labeled by PCP-mCherry (Figure 2). The 3’ UTR also contains a CAAX sequence to bind...PMC2746483 Find ribosome profiling plasmids at Addgene. 2. Halstead, James M., et al. “Translation. An RNA biosensor... determine if proteins are targeted to their destination co- or post-translationally. TRICK: Visualizing... usually translated in a specific compartment. Despite these limitations, these techniques represent powerful...

...  AAV9, AAVrg pAAV-hSyn-DIO-mCherry 37825 AAV1, 2, 5, 8, 9 (20 µL size serotype testing) pAAV-CAG-GFP...size serotype testing) pAAV-CAG-GFP 50465 AAV1, 2, 5, rg (20 µL size serotype testing) pAAV-hSyn-EGFP...best serotype for their particular study, which includes comparing the transduction patterns of different...fluorescence of the sensor. This “contrast” variant provides improved detection for tracking large populations...

...the DNA sequence (Figure 2). Accuracy and accessibility led to the widespread use of Sanger sequencing... is the genetic test used for sickle cell anemia (2). Southern blotting, in conjunction with restriction...biology98.3 (1975): 503-517. PubMed PMID: 1195397. 2. Chang, Judy C., and Yuet Wai Kan. "A sensitive new...the order of nucleotides in a string of DNA (7). Sanger sequencing uses modified nucleotides, called dideoxynucleotides...causes sickle cell anemia. The mutation happens to destroy a sequence recognized by the restriction enzyme...synthesis reaction along with all four regular nucleotides results in the synthesis of DNA strands of all...Building upon this technology, radiolabelled nucleotides were replaced with fluorescently labeled ones...

...while increasing the standard mutation rate by only ~2-fold over nCas9 levels. Two sets of modifications ...10-3 mutations per nucleotide per generation, and (2) the addition of the thioredoxin-binding domain (TBD...diversification of all nucleotides at a user-defined loci in bacteria. Its modular nature provides versatility in...provide a means to continuously diversify all nucleotides within a user-defined genomic region. EvolvR,...method developed by the Dueber Lab at Berkeley, provides a new approach for generating novel genetic variants...users to achieve targeted diversification of all nucleotides within a defined region of a genome. Require...turbidostat. Only mutate particular types of nucleotides. Both the Bassik Lab and the Chang Lab have created...

...Table 2: Lab strains of E. coli Strain Natural resistance Primary Use Genotype ccdB Survival 2 T1R ...resistance to tetracycline Additionally, Table 2 provides a quick reference for some of the popular strains...suggests there are hundreds. This only includes general lab strains designed for subcloning or protein expression...was updated on Nov 14, 2017. You've worked hard designing your plasmid – you carefully selected the optimal...Dr. Theodor Escherich, the scientist who first described them in 1885. E. coli are mainly found in the ...commercial lab strains of E. coli used today are descended from two individual isolates, the K-12 strain ...mutations found in E.coli strains Gene(s) Description Functional Consequence dam DNA adenine methylase...

...intensity of the cell’s illumination (see Figure 2). H describes the maximum signal strength, while W results...SSC-A, so most people use that setting. Figure 2: As the cell passes through the laser beam and causes...scale in proportion (while H and W do not; see Figure 2), single cell events will show up in a straight line... your cells based on their size, the cytometer provides you with two measurements: forward scatter (FSC...SSC-H, or FSC-W/SSC-W, respectively. H, A, and W describe the shape of a histogram, which graphically represents...photocurrent vs. time. The resulting histograms is described through maximum current (H), the time the cell...depends on cell type and laser voltage. The voltage describes an electric potential that can be applied to the...

...panning and scFv characterization.    Figure 2: Phage display starts with a pooled library of scFvs...the short epitope GCN4, also called a scaffold; and 2) a GCN4 binding scFv that’s fused to GFP. When the...Fv (Variable Domain) (scFv) Fragments. Antibodies 2:193–208 . https://doi.org/10.3390/antib2020193  Khantasup...recombinant antibody. They are ~25 kDa single polypeptides that contain the variable light chain (VL) and...lethal to animals.  In the clinic, scFvs’ size also provides advantages over antibodies (Ahmed et al., 2012... bond between the VL and VH domains. This bond provides stability and solubility for the scFv (Gaciarz...clinic (Bates and Power., 2019).Many of the scFvs designed for oncology applications are reformatted as bispecific...

...represent a favorable state for tumor progression (Ref 2). Future treatments may need to target this immunosuppressive...cell 30.1 (2016): 147-160. PubMed PMID: 27411589. 2. Todoric, Jelena, Laura Antonucci, and Michael Karin...positive effect on cancer treatment (Ref 1). As described in the presentation, starvation or the use of ...field of Immuno-Oncology, a sizzling hot topic, includes studies on novel cancer therapeutics such as  ... with T cell receptor engineering (Ref 5)  as described by Blue Flame Addgene depositor Professor Michel...

...sites would confound your experimental results and 2) perform targeted off-target measurements at those...experiments where only localization of the Cas occurs. 2 gRNA approach Off-targets that come from mutations...perform downstream characterization of, isolating 2 or 3 distinct clones to perform key experiments on..., it’s essential to understand them in order to design the most specific assays possible. In this blog...-target effects should impact your experimental design differently. Yes, it is always better to reduce...introducing a specified edit). However, if your design does have wiggle room, you can minimize off targets...

... you can read more about them in review articles (2–4). You can also take a look at my recent paper to...Development 118, 401–15 (1993). PubMed PMID: 8223268. 2. J. B. Duffy, GAL4 System in Drosophila : A Fly Geneticist...references and web links from my first post and start designing your first fly experiment! Many thanks to our...PMID: 28704652. 6. V. Rodríguez, D. Didiano, C. Desplan, Power tools for gene expression and clonal analysis...

...Interview with Stem Cell Researcher Darrell Kotton 2. Stem cell scientists care about patients As any grad... Yamanaka explained that it took 50 people nearly 2 years to create the first stem cell line that could...speakers from top minds in the stem cell field have described the Yamanaka paper as "life changing". Dr. Yamanaka...affordable. 4. Stem cell scientists are generous Besides some of the scientists I've already mentioned, ...

...that the signal to noise ratio can be increased from 2-fold (by CRISPRainbow) to 3-fold with CRISPR-Sirius...CNV) analysis of the U2OS cells.   Figure 2: CRISPR-Sirius tricolor labeling. (A) Diagram of sgRNA-Sirius... chromosome-specific loci in the genome and the guides are relatively unstable.   Figure 1: CRISPRainbow... the gRNA tetraloop and went through subsequent design iterations to increase stability and minimize misfolding...

...is always best to have options.      Fig. 2: Addgene’s western blot for mEGFP-HRas (from Plasmid...the presence of oxidizing agents. Adapted from Fig. 2 Campbell et al, Nature Methods   Find YFP plasmids...effects, both of happen with Cas13. CRISPR-Csm degrades both nuclear and cytoplasmic mRNA with 90-99% ...crystalizing the hfYFP structure, the group rationally designed GFP variants with similar capabilities. If you...

... as muscular dystrophies, heart disease, and type 2 diabetes, across four different types of human cells...(#186715) and hCtRNA_VT (#186716).   Fig. 2: Comparison of previous representative dual-gRNA architectures...The natural CRISPR locus of a bacteria host encodes multiple guide RNAs (gRNAs) on a single array to ...applications, we provide detailed manual and online design examples of using Addgene plasmids hCtRNA_FT (#186715...

...otherwise be difficult to separate out. In Figure 2, you can see that, without the FMO, it would be difficult... from the Ly6C positive population. Figure 2: An FMO gating example. The FMO sample (A) can be ...familiar with the basics of controls in experimental design, you’ll need a few controls specific to flow as...controls like FMO or isotype controls, which we’ll describe a bit later. Single Colors Single color controls...

...series will cover 2 of the 7 mentoring questions I set out to answer. First, I will describe some of the many...101-Building Relationships for Success”. Choose 1-2 tactics to try. One of my favorites is making the ...Watch for my upcoming mentoring post in which I describe how to organize a successful peer mentoring circle...

... you may want to consult Tables 1 and 2 in our Technical Design of a Western Blot post to make sure you... that all look exactly alike.      Figure 2: Reagent gradient. Here, the lanes are lightly marked...guide. The gel is then removed from the membrane (2) and the membrane is cut so each lane is its own piece...the antibody’s dynamic range. Specifically, the 1:2, 1:1, and No dilution samples all have approximately...conditions are 4 °C overnight or room temperature for 1–2 hours, there is some evidence that antibodies may ...right. If you’ve thoughtfully done the technical design of your blot, but are still finding yourself having...your membrane! Membranes look the same from both sides, so it’s quite easy to think lane 1 is‌ lane 8. ...

...parts: (1) β-strands 1-9 (β1-9) and the one ɑ-helix, (2) β-strand 10 (β10), and (3) β-strand 11 (β11). Each...activating caspase-3, and detected fluorescence within 2 to 5 hours, a time consistent with previous methods...fluoresce: FlipCherry. FlipCherry provides a proof of concept that the designed used in FlipGFP can be applied...range and high brightness. The Shu Lab from UCSF designed an improved apoptosis reporter, ZipGFP, which ...prevent binding, the β strands in FlipGFP were redesigned so that they are parallel to each other and thus...fluorogenic reporter toolkit, the Shu Lab also designed a red fluorogenic protease reporter based on superfolder...PMCID: PMC2117903. To, Tsz-Leung, et al. "Rational design of a GFP-based fluorogenic caspase reporter for...

...from a single Illumina MiSeq® run. As shown in Fig. 2, below, differences in input concentration across ...plexWell™ libraries is well suited to large-scale de novo assemblies of short constructs like plasmids...all over the world. The rest of this piece will describe plexWell™ in more detail, and how we are using... containing fragments carrying sample-specific barcodes. Rather than continuing to process 100s to 1000s...

...represent the preexisting resistant subpopulation 2. Resistant cells among the separate replicates would...medicine 21.5 (2015): 440-448. Pubmed PMID: 25849130. 2. Hata, Aaron N., et al. "Tumor cells can follow distinct...monitored over time by sequencing the barcodes in the population (all barcodes can be amplified using the same...with roughly 73 million semi-random, 30 bp DNA barcodes that are integrated into target cells' genomes...ClonTracer system, lentiviruses containing these barcodes infect the target cell population at low multiplicity...replicate populations, they found that similar barcodes consistently made up the majority of the resistant...resistant populations. In fact, 88% of the barcodes within the resistant populations were shared by at least...

...VP64 fusion; 2. A sgRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop 2; 3. The MS2...first generation of CRISPR-Cas9 plasmids were not designed with gene activation in mind. “We had not managed...Konermann and her colleagues in Feng Zhang’s lab describe just such a system, which they call CRISPR/Cas9...from the structure to guide and improve activator design,” she said. “Before it was a little like tapping...instead of at the protein’s C terminus. With that new design in place, Konermann immediately saw an order of...consistency and validation with >80% effectively enriched guides per gene hit, and 100% validation of the top 10...

... to share information about the emerging SARS-CoV-2 virus quickly and openly. By sharing results faster...Open Scientific Communication Ecosystem Fig. 2: The scientific ecosystem, enriched by open access...been common in fields like math and physics for decades (see: arXiv.org), but have been only adopted by...course, preprints don’t replace journals and other modes of scientific communication. Researchers often post...to receive deposits of plasmids that have been described in a preprint. In fact, over 1,500 plasmids in...Depositing plasmids that were used in a preprint provides all the same benefits as sharing those plasmids...resources on the Addgene blog Summer SciComm Series: Modes of Communication Intro to Our Summer SciComm Series...

...The groups in these programs typically consist of 1-2 mentors and 3-5 mentees who meet together for an 8... together. If you all work at the same place, a 1-2 hour lunch on a regular schedule might work. If not... for over 10 years. Many successful scientists describe having a “posse” of mentors as one key to their...Ellen Daniell. In “Every Other Thursday,” Daniell describes a group of 7 women scientists who met every other...increases the diversity of input and perspectives Provides a good mechanism for accountability that makes...leadership and communication skills. The leader also provides materials such as pre-reads, videos or exercises...

...nonsense-mediated decay. Although Nelson et al. observed only 2% genome editing in one experiment, they found the ... cells throughout the body, especially stem cells 2. Remove any immunogenicity of the AAV vector Once ...Science. 2015 Dec 31. doi:10.1126/science.aae0169 2. Long C, Amoasii L, Mireault AA, McAnally JR, Li H...dystrophin transcript. Unfortunately, the oligonucleotides only modestly improve muscle function, and they must...wasting leading to death at around 30 years of age. Despite the amount of research conducted on DMD, there ... over time, stem cell editing would be highly desirable. Should stem cell editing occur, the CRISPR components... for Duchenne muscular dystrophy. gRNAs can be designed to flank a single or multiple exons that, when...

...misplacing your results or losing that lab notebook from 2 years ago. The free subscription to Lab Archives allots...notebooks you’re saving trees! The Earth thanks you. 2. Gibco Cell Culture by Gibco Whenever I start to learn...with NEB enzymes, the app is quite helpful and provides a list of directions including which buffers to...

...immunization until the desired level of antibodies is reached - typically within 2-4 months. After the animal...animal is given additional booster immunizations every 2-3 weeks to increase the antibody titer. The titer ... animal has the desired level of antibodies in its bloodstream, the antibodies are retrieved first by ...species. Pre-adsorbed polyclonal antibodies are designed for experiments where antibodies raised in several...

.... pCRISPomyces-1 includes Cas9, a tracrRNA, and a CRISPR array, while pCRISPomyces-2 contains Cas9 and...pCRISPomyces-2 displays a higher editing efficiency, perhaps due to its condensed design. For both systems...and a subsequent curing protocol that requires only 2-3 hours incubation. E. coli and T. citrea scarless...four major components including (1) a CRISPR RNA, (2) four proteins forming the QCascade DNA-targeting ...Marraffini’s lab took advantage of this lethality to design the first synthetic bacterial CRISPR system in ...gRNA cassette for plasmid curing through "self-destruction" after induction with L-rhamnose and aTet. CRMAGE...scarless editing plasmids Sheng Yang’s lab describes a two-plasmid system that combines recombineering with ...

...and scholarships in different subject areas!    2. Resume and cover letter know-how  Tailor your resume...4. Letters of recommendation wisdom  Reach out to 2-3 individuals in your professional circle to vouch... such as citizenship status, academic year, and grades that serve as automated filters of consideration...and cover letter revised by your network, which includes your friends, family members, and professional...

...per cell increases 4-5 fold (for pUC plasmids) or ~2 fold (for p15a, R6K, and ColE2 plasmids) as cell division...biotechnology 36.4 (2018): 352. PubMed PMID: 29553576. 2. Kittleson, Joshua T., Sherine Cheung, and JChristopher...of copy number (Segall-Shapiro et al., 2018). Designing stabilized promoters To keep gene expression stable...ideally be optimized to promote generation of the desired product while avoiding the accumulation of toxic...

...without leaving behind any accessory DNA sequences [2], [3]. Simply put, the piggyBac™ transposon containing...with a Footprint-Free™ genome edit.   Figure 2: Editing DNA using the piggyBac™ transposon selection...PMID: 26211958. PubMed Central PMCID: PMC4663986. 2.  A. M. Singh, V. V Adjan Steffey, T. Yeshi, and D...facilitate the selection of cells containing your desired edit. The selection is marker is then seamlessly...sites is cytotoxic for proliferating cells and provides a simple tool for conditional cell ablation.,”...Zinc-finger nuclease-induced gene repair with oligodeoxynucleotides: wanted and unwanted target locus modifications...