We narrowed to 95 results for: Myc-tagged Cre

...different selection markers (blasticidin, neomycin, puromycin, hygromycin) and fluorescent protein reporters ... both of these cases, transposons combine the advantages of viruses and naked DNA while eliminating some... then degraded by the cell.4.) The transposase creates a double strand break in the DNA and allows the...transposase gains its name from the fact that it was recreated from a defective transposase found in fish. This...transposases. Improvements have been made since then by increasing the consensus between a greater number of extinct...primary cells. In 2009, Zsuzsanna Izsvak’s lab screened many SB tranposase variants for hyperactivity ...

...this protocol uses microscopy to determine the percentage of fluorescent cells, these cells can still be...L-alanyl-L-glutamine (or alternative stable glutamine such as glutaGRO, Corning 25-015-CI) Heat-inactivated FBS Polybrene...L-alanyl-L-glutamine (or stable alternative, such as glutaGRO) To a 500 mL bottle of DMEM high glucose, add ...55 mL of heat-inactivated FBS and 5 mL of 100X glutaGRO. Store at 4 °C. Pro-Tip Different brands and lots...obtaining accurate titers. Check the cells for mycoplasma (Link opens in a new window) regularly Do not... after 20–30 passages. Do not add penicillin/streptomycin to the media, as it can affect viability and... remove cells and debris. Lentiviral titer can decrease during cycles of freeze-thaw. If you are freezing...

...downstream of the TAM, creating staggered double-strand breaks like those created by Cas12a. Applications...plasmids contain a hygromycin resistance gene as the plant-selectable marker and a kanamycin resistance gene...TnpB from Deinococcus radiodurans (Dra2TnpB) to create a genome editing system for plants (Karmakar et... like Cas9. This is where TnpB offers a clear advantage. At approximately one-third the size of Cas9 or... proteins with additional enzymes or domains (increasing the overall size), using TnpB as the core nuclease... using your method of choice. Step 5: Colony Screening Use Primer 92F (5′-cattacgcaattggacgacaac-3′)...′) and Oligo 2 (specific to your target) to screen colonies via PCR. A 354-bp product confirms successful...

...Pooled CRISPR Libraries for Screening Use pooled CRISPR libraries to screen for genes involved in specific...carrying version 1 of the CellTag barcoding library. Version 1 of the Celltag library contains 19973 barcodes...carrying version 2 of the CellTag barcoding library. Version 2 of the Celltag library contains 4934 barcodes...carrying version 3 of the CellTag barcoding library. Version 3 of the Celltag library contains 5737 barcodes... genes along with 1000 non-targeting controls Puromycin Doench , Root 73179 Human gRNA pooled library ... genes along with 1000 non-targeting controls Puromycin Doench , Root Human activation pooled libraries...56,762 unique sgRNAs targeting 18,885 human genes Puromycin Doench , Root Human inactivation pooled libraries...

...yet they still share many of the same technical advantages that come with working with prokaryotes. This...primarily pertains to ORIs in budding yeast, Saccharomyces cerevisiae; however, we’ve also noted some features...required for the replication of fission yeast, Schizosaccharomyces pombe, vectors at the end.) In a previous...must be utilized or a new combination needs to be created in advance of the experiment. The marker provided...physiological levels due to high copy numbers. This creates a potential metabolic burden on the yeast cells...

...contains 24 copies of SunTag, a synthetic scaffold that can recruit GFP fused to the SunTag-specific antibody...Imaging by Coat protein Knock-off) instead takes advantage of the ribosome’s movement. When the ribosome ...Using translation inhibitors cycloheximide and puromycin, Halstead et al. verified that translation is ...indicating translational repression. During later stages, single RFP-labeled osk and Oskar protein were ...scFv. Each time the reporter is translated, the SunTag sites on the nascent polypeptide recruit scFV-GFP...translationally silent. Both of these methods increase our understanding of translation dynamics, but...but they come with a few caveats. In both cases, creating a reporter mRNA requires inserting multiple hairpin...

...should have a fitness advantage over proteins with single PAM recognition. Created with BioRender.com...genome-wide off-targets, since increasing PAM flexibility might also increase off-target activity. Surprisingly...gone one step further, using directed evolution to create xCas9, an enzyme recognizing a broad range of PAMs...PAMs like NG, GAA, and GAT, but also displaying increased editing specificity. We’re excited to learn more...easy, right? Yes and no. If you’re just trying to create a knockout, you might be happy targeting anywhere...precise edits or knock-ins. Editing efficiency decreases drastically once your cut site is more than 10...insertion site. Base editing, another method to create point mutations, also has a strict editing window...

...Wyatt and Dale Ramsden, UNC at Chapel Hill. One advantage to using the CRISPR/Cas system for genome engineering...Persistence of a Chromosome Break Diminishes Its Mutagenic Potential. PLoS Genet 5:e1000683 . https://doi.org...End-Joining Strategy for Efficient Genome Editing in Mycobacterium tuberculosis. mBio 11: . https://doi.org/10.1128...Genome-Wide Control for Large-Scale Functional Screens        ...

... in yeast Base editing CRISPRi in Caulobacter crescentus Multiplexed gRNA for human pluripotent stem cells...that controls repression of gene expression in Saccharomyces cerevisiae. The system consists of dCas9, the...editing. Using several rapid and cost-effective screening methods, David Liu’s lab identified and validated... base editor plasmids CRISPRi in Caulobacter crescentus Figure 1: Serial dilutions of CRISPRi in...adapted from Guzzo et al., 2020. Caulobacter crescentus is a popular model organism for studying the ...ctrA sgRNA and saw that the levels of ctrA mRNA decreased after 20 minutes in xylose (which controls expression... 2: The components of Casilio-ME1. Image from Taghbalout et al., 2019. Fusing epigenetic modifiers...

...Figure 2: CRISPR R-loop. Created with BioRender.com. Base editors take advantage of this single-stranded...nucleobase component of the nucleoside inosine. Created with BioRender.com. Although it was a huge ...have scientists solved this problem? By taking advantage of repair pathways within the cell — in some cases...it finds a PAM, Cas9 unwinds the adjacent DNA to create a region of separate strands called an R-loop (...activity window restricted to the stretch of ssDNA created by Cas9 binding. Pro tip! In CRISPR, the non-target...BER process was excising the incorrect U base, creating an “abasic,” or empty, DNA site, and then replacing...in the same journal on the same day.) This team created a similar construct that they called a Glycosylase...

...the human gut bacteria B. thetaiotaomicron, Mycobacterium, Pseudomonas, and E. coli. However, many non-model...for CRISPRi transfer Mobile-CRISPRi offers two advantages over existing bacterial CRISPRi methods. First...better suited for genome-wide CRISPRi library screening experiments where maintaining library diversity...of antibiotics are often essential genes which creates a challenge when studying them since their complete...

...DNA end of the break is resected by nucleases to create a ssDNA 3’ overhang. This will serve as both a ...considerations for designing a repair template to create mutations HDR donors can contain insertable sequences...circular plasmid templates is generally low; to increase the frequency of edits, researchers have designed...complementary ssDNA. Easi-CRISPR was shown to increasing editing efficiency from 1-10% with dsDNA to 25... chromosomal aberrations. Pfeiffer P, et al. Mutagenesis (2000). PubMed. Multiple Pathways of Recombination...Recombination Induced by Double-Strand Breaks in Saccharomyces cerevisiae. Paques F and Haber JE. Microbiology...Frequency of Homology-directed Repair (HDR) for Increased Efficiency of CRISPR/Cas-mediated Transgenesis...

...majority of plasmids contain either an ampicillin or kanamycin resistance gene. Every step along the way involves...and prepare fresh media with antibiotics at each stage. The barcodes and LIMS help us track each sample...deposit. Using barcoded tubes is part of Addgene’s secret to success. It helps us quickly process samples...

...promoters and create sophisticated patterns of UAS-driven gene expression. To further increase the complexity...system into a repressible system, Gossen and Bujard created the tetracycline-controlled transactivator (tTA...bacterial operons. A chimeric transactivator, cTA, was created by fusing CymR to the activation domain VP16. cTA...GAL4 gene downstream of a tissue- or developmental stage-specific promoter and design a UAS reporter construct...state, and co-expression of the inducer QF can increase QUAS driven expression by ~3,300-fold in Drosophila...genes for metabolic engineering applications in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197...

...transgene expression. Popular uses include Cre-lox mice, knockouts using CRISPR or shRNA , and overexpressing...removed or mutated for increased safety. Figure 1: Wild-type lentivirus genome. Created with BioRender.com...in-stock lentiviral preps . CRISPR Genomic Screens Genome-wide screens are an efficient way to determine what... use the packaging genes gag , pol , and env to create viral particles. Wild-type lentiviruses additionally...replication-competent lentivirus production — that is, creation of viral particles that could infect cells and...Figure 2: First-generation lentiviral plasmids. Created with BioRender.com. Second-generation Second-generation...Figure 3: Second-generation lentiviral plasmids. Created with BioRender.com. Third-generation The third-...