We narrowed to 965 results for: REP

... used as a template for repair? Read on to find out! Homology-directed repair and genome engineering The...is to utilize the built-in repair pathway cells already have for repairing DNA double strand breaks – ...homology-directed repair (HDR) pathway in mammalian cells, but a less frequent DNA repair mechanism in general...suggestions. Disable the other repair mechanisms Mammalian cells have 2 primary repair mechanisms besides HR -...and tricks for harnessing the homology-directed repair pathway, designing the best donor DNA, and avoiding...a frameshift mutation. The majority of cellular repair events will generate these types of mutations, ...the cell to use the donor DNA as a template for repair, which will result in the incorporation of your...

... vector preps!   Figure 1: A) Cumulative AAV preps available in the Addgene repository each year...Cumulative AAV preps available at Addgene from 2016–2023 by type of tool. B) AAV preps distributed by ...AAV preps for the seven most popular serotypes available from Addgene from 2016–2023. B) AAV preps for...jGCaMP7, and jGCaMP8 viral preps distributed per year compared to other AAVs preps from 2016–2023. B) Inset...prioritizing which preps to include in our service allows us to make ready-to-use viral preps that are available...been finding and sharing stories unique to our repository and resources. As editor of Addgene’s eBooks ...both to produce and to verify the quality of the prep. This was a large factor in Addgene’s decision to...

...involve repairing chromosomal DSBs. It is important to know that cells possess two major repair pathways... has a higher capacity for repair, as there is no requirement for a repair template (sister chromatid ...introduced breaks are repaired. The following factors are required for NHEJ repair regardless of end structure...introduced. Repair of Cas9-induced breaks by NHEJ As illustrated below, NHEJ-mediated repair of Cas9-generated...accounting for less than 5% of repair events. However, the products of accurate repair are easily re-cleaved while...Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR) – and how these pathways work, as this could... or extensive DNA synthesis. NHEJ also finishes repair of most types of breaks in tens of minutes – an...

... even more powerful following a report that one can use linear repair templates (ie. PCR-derived dsDNA...single-stranded repair templates (Farboud et al., 2019).   The Mello lab also reported that modifying ... 2014). A further development of this approach reported in 2017 uses the direct injection of a CRISPR/...Cas9 ribonucleoprotein complex with linear DNA as repair templates which reduces time to generate edits ... I developed a co-selection approach relying on repair of a conditional-lethal mutation to identify edits...edits with minimal screening effort. Selecting for repair of a lethal mutation should remove much of the ...lethal mutation survive the selection; from a representative co-selection experiment, five knock-ins were...

...genes as reporters and some that repress instead of activate transcription. The HIS3 reporter system (...the promoter of the reporter gene to activate expression (Figure 2c). The reporter gene then allows detection...positives: Run replicates of the experiment to reduce the likelihood of indiscriminate reporter gene activation...sequence (UAS) of its target gene, in this case a reporter gene (e.g. luciferase or GFP), regardless of whether...interacts with a transcriptional activator (1a) or a repressor (1b). Scientists took advantage of these discoveries...co-expressed in a yeast strain that also contains a reporter gene cloned downstream of a promoter containing...referred to as the “bait”, Figure 2b) cannot activate reporter gene expression on its own, but, if the two POI...

...polycistronic cassettes and discovered reprogramming discrepancies, which they traced to a 9 amino acid ...of Klf4 did not lead to the observed reprogramming discrepancies. The polycistronic piggyBac transposon...SOX2 acts to reprogram human fibroblasts into induced pluripotent stem cells. By replacing wild type SOX2...the efficiency of iPSC reprogramming while the HP1 fusion eliminates reprogramming. These results argue ...highlight a subset of the new plasmids in the repository through our hot plasmids articles. These brief...posts for every great plasmid that comes into the repository, but be sure to let us know if you'd like to ...KLF4, and c-MYC is the established model for reprogramming somatic cells. A common method for generating...

...of clones   lacIq lac repressor overproduced, expression from pLac repressed more   LacZ β-galactosidase...that carried out the labor-intensive process of replicating your new plasmid: the Escherichia coli bacteria...cloning, routine cloning, cloning unstable DNA, preparing unmethylated DNA, and more. Many mutations that...Consequence dam DNA adenine methylase mutation (GATC) Preparing unmethylated DNA, important when trying to cut... dcm DNA cytosine methylase mutation (CCWGG) Preparing unmethylated DNA, important when trying to cut... is essential for recombination and general DNA repair Reduces plasmid recombination, increases plasmid...subunit S12 of 30S ribosome Confers resistance to streptomycin Tn10   Confers resistance to tetracycline ...

...performing different sized preps, such as miniprep, midiprep, etc. (more on prep sizes below). But what do...Standard Prep Kit Capacities* Prep size Amount of culture Approximate DNA yield** Miniprep 1–5 mL ...mL 5–50 µg Midiprep 25–100 mL 20–200 µg Maxiprep 100–500 mL 100–750 µg Megaprep 0.5–2.5 L 1.5–2.5...you choose the plasmid prep kit that is right for you. What type of plasmid prep do you need? As we covered...covered in a previous post on plasmid preps, not all plasmid preps are the same. Your downstream application...and you can get away with slightly lower quality preps, while other applications require large amounts ...quality and quantity.   Figure 1: Plasmid preps fall into different categories based on their relative...

...pleased to report that over 95% of viral capsids in representative viral vector preparations are genome-containing...vector preparations. Addgene has used this method to generally validate our viral vector preparation protocol... people usually report? Addgene and other viral vector manufacturing facilities report the physical titer...precise and more repeatable than qPCR (Taylor et al., 2017). However, because purified AAV preps contain similar...cell transduction assays. Wild-type AAV2 has been reported to have a near-perfect physical-to-infectious ... However, for recombinant AAV2, the same study reported a physical-to-infectious particle ratio of 50:...al., 2010). The specific infectivity of viral preparations is defined by the ratio of physical viral particles...

...many different experimental procedures, such as minipreps and RNA extractions. Plus, centrifuges are useful... are separating solids from a liquid, pellets represent the solids in your sample while the liquid portion...

...General protein expression  m15 pREP4*, ** Kanamycin (pREP4) Cis-repression of the E. coli T5 promoter (found... (lac repressor on the pREP4 plasmid). Expression vector cannot have p15A origin of replication F-, Φ80ΔlacM15...as M15(pREP4) use a lac repressor to act directly on the expression plasmid in order to repress transcription...expression vector cannot have p15A origin of replication F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) pLysS...expression vector cannot have p15A origin of replication F- ompT lon hsdSB (rB- mB-) gal dcm (DE3) pLysE...Tetracycline RecA-deficient; best for plasmids with repetative sequences.  F- ompT lon hsdSB (rB- mB-) gal ...toxic or insoluble proteins Origami2 (DE3)** Streptomycin and Tetracycline Contains highly active thioredoxin...

... offering virion preparations to the scientific community. Addgene's AAV preparations have worked well...lentiviral preps for the delivery of Cas9 endonuclease, Cas9 nickase, Cas9 activators, Cas9 repressors, select...money producing virus from select vectors in the repository yourself, you can now order ready-to-use virus...lentivirus (with many CRISPR tools included among the preps that are currently available) and adeno associated...with optogenetic tools coming soon). The viral preparations undergo rigorous quality control testing at ...faster by producing, titering, and testing viral preps for them. At the same time, we’re developing ourselves...scientists and accelerate research." What viral preps are available now? There are a wide variety of tools...

... own machinery to replicate the viral genome and proteins. This is, for the intrepid researcher looking...that in a bit! Viral genomes and replication When it comes to replication, viruses tend to march to the ...methods of replication. In fact, an entire classification scheme, known as the Baltimore replication classes...developed around how they replicate (Figure 2). Figure 2: Baltimore replication classes. Image credit: ...an intermediary DNA replication step between DNA and mRNA. DNA genome replication can involve an RNA or...before being translated into proteins. RNA viruses replicate their genomes using a variety of RNA or DNA intermediaries... bacteria and eukaryotes, which use a binary replication process whereby one cell copies itself and “splits...

...streak for single colonies, sub-culture, and prep with a DNA prep kit or your lab's favorite in-house protocol...plasmid yields from your prep, try our tips below! How to optimize your plasmid prep We'll discuss the following...for plasmids with repeat elements, such as viral plasmids with inverted terminal repeats. We use NEB Stable...plasmid DNA and lead to degradation of your prep. Many plasmid prep kits recommend performing an extra wash...How to Get Better Plasmid Midiprep Yields BitesizeBio: Better Plasmid Midipreps Part II: What Causes Low...updated on March 21, 2018. Most of the time, plasmid prepping is a breeze. You get your stab from Addgene, streak... multiple techniques for a particularly stubborn prep. Check the bacterial strain Increase the volume...

... the 3’ UTR of the reporter mRNA via an MS2 coat protein (NLS-MS2-RFP) the reporter mRNA, which contains...development, the osk-TRICK reporter was double labeled, indicating translational repression. During later stages...proteins to allow translation to occur. For a TRICK reporter mRNA this displacement leads to a change in the...GFP protein that binds to the coding region of a reporter mRNA via a PP7 coat protein (NLS-PCP-GFP) an RFP...translation, both GFP and RFP are bound to the reporter, causing it to appear yellow. The act of translation...of the mRNA, leaving only a red FP bound to the reporter. Because it contains a nuclear localization signal...copies of the FP-coat protein fusions to their reporter mRNAs. Using translation inhibitors cycloheximide...

...are used across species: replication, selection, and expression. Replication It’s usually not enough to...chromosome has a single origin of replication (ori), where DNA replication begins, so a bacterial plasmid...vector with a reporter, such as a fluorescent protein, to visualize vector uptake. These reporters might be...fast-growing populations of cells, the vector needs to replicate alongside its host, or it will be lost from the...compatible ori that will be recognized by its host’s replication machinery. A shuttle vector designed for unrelated...complicated. Yeast vectors may contain an autonomously replicating sequence (ARS), a partial centromere sequence...experiment. Yeast shuttle vectors have these replication sequences for their yeast targets as well as ...

...trans-acting RepA protein under the control of IPTG. Because RepA is a plasmid replication initiator protein...genes closer to the origin of replication will be overrepresented compared to those further away. As a...increases, the amount of repressor protein also increases thereby intensifying repression of the gene of interest...problems. Rapidly dividing cells initiate genome replication more than once per cell division and thus, genes...regulation by using a promoter that is responsive to a repressor protein, the increase in expression due to copy...Segall-Shapiro et al., they chose a TALE protein as the repressor because TALEs can be programmed to bind tightly...sequences and have been shown to achieve ~100 fold repression in E. coli. In addition, TALEs bind non-cooperatively...

...taking advantage of repair pathways within the cell — in some cases, the same repair pathways that were...an —NH2 (amine) group from the target base and replaced it with a double-bonded oxygen. This simple enzymatic...calling it the “edited strand.” Uracil excision repair leads to the first base transversion editor The...most CBEs include an inhibitor of base excision repair (BER), the cellular process that catches mutated...creating an “abasic,” or empty, DNA site, and then replacing the incorrect base with a G. Pro tip! Abasic sites...even better cytosine transversion editors. New DNA repair proteins fused to CGBEs Instead of using UNG, the...fusions of a CBE with other BER components: the DNA repair protein rXRCC1, or the DNA binding and lyase domain...

...mismatch repair (MMR) system. MMR’s job is to correct DNA mismatches that occur during DNA replication. There... and repressor CI. For recombineering purposes, a temperature sensitive version of the repressor gene,...lowering the temperature allows the repressor to re-nature and again repress expression of exo, beta and gam...Overview of using Lambda Red recombineering system to replace a gene of interest with an antibiotic resistance...as non-selectable DNA fragments, such as gene replacements and tags.  The typical frequency of recombinants...targeted, it’s best to determine the direction of replication through your region of interest and design an...it’s easier to not determine the direction of replication and instead design oligos that target both strands...

...been designed by the Addgene nonprofit plasmid repository and is being shared outside of Addgene for informational...informational purposes only. If you choose to reuse or repurpose this SOP in another location, please note that...