... 2. Replicates and repeats—what is the difference and is it significant? Vaux DL, et al. EMBO Rep. Apr...same aliquot/preparation. The replicate control isn’t measuring or assessing reproducibility of the effect...independent variable. Replicate controls: technical and biological Reliable results are reproducible from properly...two types of replicate controls: technical and biological. In general, technical replicates can be thought...Conversely, biological replicates ARE independent and can be thought of as "reproducibility controls". Biological...several different days. Biological replicates control for the reproducibility and consistency of the observed... employ transfection, negative, positive, and replicate controls.  Transfection controls: empty vector...

...Saito K, Ohki R, Fujii H. Sci Rep. 2013 Nov 8;3:3171. doi: 10.1038/srep03171. Efficient isolation of specific..., as well as the Editorial Board of Scientific Reports and PLoS One. Additionally, you are involved in...

...allow for replication. However, if plasmids have their own replication machinery such as Rep or other ...ori2, ori3) and replication initiation proteins (RepA, RepB) necessary for replication in various hosts... and W are known to have replicons with broad host ranges. Popular replicons from these groups include...different hosts that a plasmid can transfer to, replicate in, and persist in. Some plasmids have a narrow... while others have a broad host range and can replicate and stably maintain the genes they carry among.... These features are: The plasmid origin of replication (ori) contains structural elements that are versatile...structure allows plasmids to utilize diverse host replication machinery. For instance, the ori typically contains...

...compact expression cassettes. Sci Rep 5: . https://doi.org/10.1038/srep10777  Garneau JE, Dupuis M-È, Villion...CRISPR-based RNA editing, the REPAIR (RNA Editing for Programmable A to I Replacement), system is also small ...infection of certain tissues. However, packaging Streptococcus pyogenes (SpCas9) and a gRNA together (~4.2 ...Cas9 include the use of St1Cas9 (~3.3 kb) from Streptococcus thermophilus and a rationally-designed truncated...direct in situ breaks labeling, enrichment on streptavidin and next-generation sequencing). Using this ...

...longer single-stranded DNA.” Sci Rep. 5 (2015):12799. DOI: 10.1038/srep12799 PubMed PMID: 26242611 PubMed ...ll design your repair template with 55-100 bp homology on each side. For optimal repair, place the loxP... of ssDNA donor molecules for homology directed repair. Using long ssDNA donors, the Gurumurthy and Ohtsuka...suffer from low efficiency when a standard dsDNA repair template is used. The original design for creating...two gRNAs targeting two intronic regions and two repair templates in order to insert two loxP sites. Since...including one loxP site insertion and one NHEJ-repaired indel, often occur. Figure 1: In conventional...courtesy of Wenning Qin. Single-stranded DNA repair templates Short single-stranded DNA templates have...

...Epithelial Cells Generated from Patient iPSCs.,” Cell Rep., vol. 12, no. 9, pp. 1385–90, Aug. 2015. PubMed ...marker is included in a standard homology directed repair template to facilitate the selection of cells containing...donor is used by the cell’s host machinery for DNA repair by homologous recombination, which results in the... fact that PBx relies on the inverted terminal repeats (ITRs) found on the piggyBac™ donor for its activity... K. Schwarz, “Zinc-finger nuclease-induced gene repair with oligodeoxynucleotides: wanted and unwanted... with FLEx Vectors  Learn how Homology Directed Repair Is Used for Genome Editing Resources on Addgene.org...

...viral vectors: packaging capacity and the role of rep proteins in vector purity." Human gene therapy 18.2...rate limiting step and can immediately undergo replication and transcription. However, with scAAV the effective... complexes to study neural function. Often the reporter proteins associated with these complexes are so...cell populations. Find Ready-to-Use Retrograde AAV Preps at Addgene 2) Comparison of genetically engineered... Vectors for Your Research Get Ready-to-Use AAV Preps Browse All of Our Viral Plasmids and Resources ...

...optimized. Plasmids for transfection: pHelper; pRC (Rep-Cap), plasmid expressing your gene of interest Triton...handled carefully. Avoid touching the cells when replacing media. T-175 flask, Corning 430825, 175 cm 2 Cellstack...mM Tris HCl, 150 mM NaCl, 2 mM MgCl2) Reagent Preparation DMEM Complete : 10% v/v FBS and 4 mM L-alanyl-L-glutamine...et al., 2018 (Link opens in a new window) ). To prepare D1 + 0.1 M sorbitol, add 57 mL of 1.46 M sorbitol...bp) DNA Concentration (μg/μL) Volume of DNA (μL) RepCap 7,265 1.00 727.6 pHelper 11,854 1.00 1,185 Transfer..., for each plasmid we need: Sample Calculations RepCap: 0.08 μg/bp × 7,265 bp = 582.1 μg Volume Needed... toward the cap. If the media touches the cap, replace it with a new one before putting the CS5 in the...

... really great signs. The Congressman for Albany, Rep. Paul Tonko also came and spoke in support of science...Disclaimer: The views represented below are those of the writers and do not necessarily represent the views of ...for our public policies—many of which impact reproductive rights and justice. Whether it’s used to fight...misinformation and propaganda that limits access to full reproductive health care. And it’s needed most in low-income...According to the Guttmacher Institute, most women of reproductive age live in a state with at least two abortion...conflict with scientific evidence. We need representatives and policymakers who not only believe in the...

...CRMAGE: CRISPR Optimized MAGE Recombineering." Sci Rep. 6:19452. PubMed PMID: 26797514 Find plasmids from... by two CRISPR repeats. The spacer can be digested using BsaI, allowing a spacer-repeat “brick” to be ...include humanized wt Cas9, dCas9 (transcriptional repressor), and dCas9-VP64 (transcriptional activator)-containing...nickase mutant (Cas9n), dCas9 (transcriptional repression), and Fok1-dCas9 (dimeric nuclease). Gateway ...plasmids, which contain various promoters and reporters, and subsequently inserted into a Cas9-containing...CRISPR arrays for dCas9-based transcriptional repression. The CRISPathBrick plasmid contains a nontargeting...were to develop an array using 3 distinct spacer-repeats, you could easily create 7 unique arrays (e.g. ...

...Optimized MAGE Recombineering. Sci Rep 6: . https://doi.org/10.1038/srep19452 Selle K, Fletcher JR, Tuson H...end-joining (NHEJ) repair pathway is not very robust. In many cases, homology-directed repair does not function...oligonucleotide repair template. Through recombineering, the locus of interest is modified to match the repair template...curing time. pCRISPomyces for editing in Streptomyces  Streptomyces bacteria produce a wide variety of bioactive...elements, like a repair template, using Gibson Assembly or restriction enzyme cloning. Streptomyces bacteria ...success of Cas9 mediated repression in the Gram-positive bacterium, Streptococcus pneumoniae. Robert Husson...pTargetF contains the specific gRNA(s), and the repair template is supplied as a dsDNA fragment. Each ...

... in the associated publication. Ma, et al. Cell Rep. 2015. PubMed PMID: 26190106 PRESTO-TANGO: An open-source...hard-to-study cell types. Natsume T, et al. Cell Rep. 2016. PubMed PMID: 27052166 Nishimura K, et al. ...native methyl-directed mismatch repair (MMR) system needs to be repressed in the host strain. To address...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 ...growing number of CRISPR libraries from Addgene’s repository that can be used to perform screens in human ...-ome using a quick, affordable, and ubiquitous reporter assay. In their recent publication, Kroeze et ...

...sequence. The resulting DSB is then repaired by one of two general repair pathways: the efficient but error-prone...directed repair (HDR) pathway. HDR relies on copying DNA from a matching template to accurately repair or fill... NHEJ is a naturally occurring repair pathway and is the primary repair mechanism for DSBs. While this...base excision repair pathway, which fully removes the uridine from the DNA and replaces it with another... tool to activate, repress, visualize, and isolate genes. Activation or Repression of Target Genes Figure...sufficient to repress transcription by blocking initiation. More robust transcriptional repression (CRISPR ...demethylation DNMT3A - repression through cytosine methylation MQ1 - repression through cytosine methylation...

...Tagging Purification Controlled Expression Reporter Plasmids Reporter Plasmids Tagging and Visualization Purification...Plasmids pBAD (arabinose inducible, glucose repressible) His, Strep, mOCR, MBP, with and without SUMO or TEV...expression at a specific level. Return to Top Reporter Plasmids Reporter plasmids can be used to detect events...they activate expression of the reporter gene. Plasmid ID Promoter Reporter PI Purpose pRU Series Various...Purification Controlled Expression Reporter Plasmids You may also like… Plasmids 101 Addgene's Molecular...Bacteria? E. coli are commonly used to store and replicate plasmids of all types, but beyond that, researchers...Plasmids T7-lacO (lactose/IPTG inducible) Flag, His, Strep, mOCR, MBP, GFP, with and without SUMO or TEV cleavage...

...a maxi-prep culture. Prep size may be scaled based on the required DNA yield. Perform maxi-prep for each...colonies and inoculate into a mini-prep culture. Perform mini-prep for each sample and sequence each colony...Blog Posts Addgene is proud to present a video reprint from the Journal of Visualized Experiments (JoVE...clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system may be...DNA segment by non-homologous end joining (NHEJ) repair. Deletions have potential advantages as compared...oligos at a concentration of 100 μM in ddH 2 O. Prepare a 10 μl reaction mix for each guide and its reverse...using a Golden Gate assembly cloning strategy 3 . Prepare a 50 μl reaction mix: 100 ng circular pX330 vector...

...pcDNA3.1(+)_Strep-Lrrk2-Myc LRRK2 Myc, Strep CMV Parkinson's Maik Hintze 161583 pPuro3.1(+)_Strep-Lrrk2-Myc...Myc, Strep CMV Parkinson's Maik Hintze 161584 pPuro3.1(+)_Strep-Lrrk2(D1994S)-Myc LRRK2 Myc, Strep CMV ...pPuro3.1(+)_Strep-Lrrk2(G2019S)-Myc LRRK2 Myc, Strep CMV Parkinson's Maik Hintze 161586 pPuro3.1(+)_Strep-Lrrk2...A11(2-196)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196209 GB1-A11(2-196, G38R)-Strep ANXA11 His... His, Strep, Tev T7 ALS Lalit Deshmukh 196210 GB1-A11(2-196, D40G)-Strep ANXA11 His, Strep, Tev T7 ALS... G175R)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit Deshmukh 196212 GB1-A11(2-196, G189E)-Strep ANXA11 His... His, Strep, Tev T7 ALS Lalit Deshmukh 196213 GB1-A11(2-52)-Strep ANXA11 His, Strep, Tev T7 ALS Lalit ...

...While our repository serves the needs of many researchers, scientists sometimes require preps beyond our...service with all-in-one pricing for viral vector preps, which includes MTA facilitation, DNA amplification...protection levels. We offer 963 in-stock viral vector preps, delivering high quality and ready availability ...start experiments as soon as they receive their AAV prep from Addgene — eliminating the steps to produce,...recombinase, and biosensor transfer plasmids in our repository are eligible for this service. Select any eligible...eligible plasmid in the Addgene repository in your choice of: serotype (AAV1, AAV2, AAV5, AAV8, or AAVrg...ordered in three sizes. For all viral vector preparations, we set both minimum standards and typical titers...

...pTETRIS-cargo Firefly For use as a reporter of the ability of a given lncRNA to cis-repress Mauro Calabrese 106253...Multiple Multi-luciferase reporter vector including transcriptional reporters for NF-kb, TGF-b, c-Myc, ...transcriptional regulation and bioluminescent reporters. Plasmid...Highlights Empty Backbones Expression Constructs Reporter Constructs Luciferase is the enzyme responsible... common choice in assays ranging from use as a reporter gene in vitro and in vivo to use in biosensors...-lives, making them useful as transcriptional reporters. However, they are not secreted, so cells must...CMV-LUC2CP/intron/ARE, CMV-LUC2CP/ARE : A splicing reporter that contains an intron in the middle of the Firefly...

... RNA editing, gene replacement therapy, RNA trans-splicing, and protein replacement to reverse the impacts...suggested roles in transcriptional repression, activity dependent de-repression, chromatin remodeling, gene ...Cell Line Repositories (Link opens in a new window) Coriell Institute - cell line repository, including...in a new window) MMRRC Repository - NIH-supported mouse and cell line repository (Link opens in a new window...largest biorepositories (Link opens in a new window) ATCC - the world's largest cell line repository Tissue...spoken language and hand skills, development of repetitive hand stereotypies, and gait dysfunction or absence...to bind to methylated DNA and recruit known co-repressors or other transcriptional modifiers suggests the...

.... When a repair template is present, the cell may repair a DSB using homology-directed repair (HDR) instead...be used with a repair template to introduce specific edits via homology-directed repair (HDR). ID Plasmid...gRNA-defined target sequence. DSBs are preferentially repaired in the cell by non-homologous end joining (NHEJ...genomic changes, researchers use ssDNA or dsDNA repair templates with homology to the DNA flanking the...Zebrafish Xenopus CRISPR Resources CRISPR Guide Viral Preps Protocols gRNA Design Tools CRISPR Blog Posts Base...base editors convert adenine to inosine, which is replaced by guanosine to create A->G (or T->C on the opposite...nicks create a double-strand break (DSB) that is repaired using error-prone non-homologous end joining (...