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We narrowed to 956 results for: REP

Showing: 281 - 300 of 956 results

  1. CRISPR 101: RNA Editing with Cas13

    Type
    Blog Post
    ...This new system, termed REPAIR (RNA editing for programmable A to I (G) replacement) is the first CRISPR ...RNA editing from the Cas13b REPAIR system To test the robustness of REPAIRv1, the lab targeted 34 pathogenic...transcriptome-wide comparison, REPAIRv2 reduced off-target sites from the 18,385 observed with REPAIRv1 to merely 20. ..., RNA editing doesn't require homology-directed repair (HDR) machinery, and could thus be used in non-... 30-84 nucleotides, and designated this system REPAIRv1. Using next-generation sequencing, they confirmed...HEK293T cells, as assessed using RNA-seq. Since the REPAIRv1 machinery is too large to fit into adeno-associated...identified ADAR2DD(delta984-1090), which decreases the REPAIRv1 construct size from 4,473 bp to 4,152 bp without...

  2. Learning Technical Skills at a Lab Central Workshop

    Type
    Blog Post
    ...part of my job, in addition to preparing DNA and generating/replenishing kits, is to find new ways to make...scale DNA preps with multiple samples without cross contamination. We currently use the CosMCPrep system ... throughput DNA prepping by trying out NEB’s new NEBNext Ultra II FS DNA Library Prep Kit for Illumina...of this newly released prep kit. In their prior NEBNext Ultra II DNA Library Prep Kit for Illumina, one... process for library preparation using this kit is: Fragmentation and end repair Adapter Ligation Clean...advancement easier. NEBNext Ultra FS DNA Library Prep Kit To create a DNA library, one must first fragment...mostly used to make new libraries as opposed to replicating them like we do at Addgene), going to the workshop...

  3. Antibodies 101: The Four ELISAs and When to Use Them

    Type
    Blog Post
    ...conjugated with a reporter or tag that can then be detected.  Typically, the reporter is an enzyme such...dish and incubate it with a reporter conjugated primary antibody. The reporter reacts with a provided substrate...simplicity we will discuss the more common enzyme-based reporter methods. For enzymatic signal detection, users... sandwich and competitive. In a direct ELISA a reporter enzyme conjugated antibody binds to antigen coated...antibody first binds to the antigen and then a reporter enzyme conjugated secondary antibody binds to ...detection antibody can be directly conjugated with a reporter enzyme or a conjugated secondary antibody can ...).  The sample (purple) is preincubated with a reporter enzyme labeled antibody and then added to the ...

  4. Hot Plasmids - August 2020

    Type
    Blog Post
    ...a subset of the new plasmids and viral preps in the repository through our hot plasmids articles. These...dCas-VPR repressor are introduced into the hPSC genome using AAV1 and contain an EGFP reporter. Expression...to provide ready-to-use viral preps. Here are some of the new viral preps from recent months: New GPCR...measurements of norepinephrine. These norepinephrine sensors include GRAB_NE1h (high affinity) and GRAB_NE1m...production in E.coli as simple as following a DNA miniprep protocol!  First, the authors created the pMAK...Expression of the dCas9-KRAB activator or dCas9-VPR repressor in hPSCs is also driven by a doxycycline inducible...promoter. The gRNA piggyBac plasmids include a mRFP reporter. With this system, they could quantify and track...

  5. The Challenges of Cell Culture

    Type
    Blog Post
    ...validation carried out by the commercial repository. Repositories generally ensure cell line identity and...microbiology, and pharmacology (many of which are represented in Addgene's Special Collections). Arguably, ...invariably much older than those available from biorepositories leading to genotypic and phenotypic artefacts...interpretation in cell culture for this reason. Unreproducible results are rife. Misidentified or cross-contaminated...been known about since 1966 when Stanley Gartler reported that most of the 18 cell lines he had studied ...easier to conduct high quality cell culture. Biorepositories around the world ensure that the cell lines...The oldest, largest, and most diverse of these repositories is the American Type Culture Collection aka ...

  6. Prime Editing: Adding Precision and Flexibility to CRISPR Editing

    Type
    Blog Post
    ...more efficient than homology-directed repair Homology-directed repair (HDR) stimulated by double strand breaks...during mismatch repair   ✓   ✓ ~ ~ ~ Transiently co-express mismatch repair inhibitor MLH1dn...originally published in Nature, is a “search-and-replace” genome editing technique that mediates targeted...heteroduplex is resolved by the cell’s mismatch repair system, and more recent prime editing innovations...innovations have improved the odds of mismatch repair favoring the edited strand. The first iteration of prime.... We’ll summarize the major ones in Addgene’s repository later in the post. Advantages of prime editing...relatively low, meaning that most Cas9-induced DSBs are repaired by non-homologous end joining. As a result, Cas9...

  7. A Control for All Seasons

    Type
    Blog Post
    ...biological replicates such as multiple mice in each group and C, technical replicates or repeated measurements...results.  Replicates Replicates are another critical control in biological experiments. Replicates can be...statistical power of an experiment. Technical replicates are repeated measurements of the same sample and are...experimental protocol. In the experiment above, you could repeat the tau western blot several times with the same...difficult to obtain meaningful results.  Biological replicates are distinct samples that are treated identically... on the results of a single mouse. Biological replicates help you to determine which responses arise from...will be used (Burry 2011). It does not need to be repeated for each experimental run as long as the protocol...

  8. AAV Vector Quality Control: Going the Extra Mile with NGS

    Type
    Blog Post
    ... Meghan Rego. Reproducible data are key to science, so scientists are used to repeating experiments to...the plasmid used to create the viral prep. In a clean viral prep, like AAV-44362 shown below, more than...confirm their findings. But no scientist wants to repeat an experiment because of poor reagent quality. ...the wrong plasmid? What if bacteria snuck into the prep during the production process? These questions are...contamination can be identified, and we will discard any AAV prep we suspect is contaminated. For AAV-68544, the best...method we wanted to see how low we could go. We prepared a range of spiked samples from 0.1-20% and discovered... detection software confirms the serotype of the prep by interrogating the VGS data for small predetermined...

  9. RNA Interference in Plant Biology: New Tools for an Old Favorite

    Type
    Blog Post
    ...fluorescent reporter, directly couple silencing of your gene target to silencing of the reporter. This is...target sequence against the fluorescent reporter in an inverted repeat orientation. Therefore, a single hairpin...Figure 1). The plant kingdom has expanded its repertoire of Dicer enzymes and these different Dicer-like...desired target of RNAi is expressed as inverted repeats separated by a loop region that facilitates the...conclusion was based upon numerous phenotype discrepancies between gene silencing and knockout methods ... complementary to your gene target as inverted repeats (Figure 1). With this technique, multiple genes...fusing your gene target sequence to a fluorescent reporter sequence (discussed below). Examples of these ...

  10. Analyzing CRISPR Editing Results with ICE from Synthego

    Type
    Blog Post
    ...their relative representation in the edited pool. The black vertical dotted line represents the cut site...vertical black dotted line represents the cut site. Cutting and error-prone repair typically result in mixed...also provides a Knockout score (KO-score) which represents the proportion of cells that have either a frameshift...For a multiplex sample, ICE includes visual representations of all detected edit types in the sample. The...complete, a new screen appears with a graphical representation of the results and a list of the analyzed samples...used. Currently, ICE is configured for Cas9 fromStreptococcus pyogenes (SpCas9). ICE Score - The editing...correlation coefficient (r) is also computed and reported.  The higher the R2 value, the more confident ...

  11. Rett Syndrome: A History of Research and Therapeutic Outlooks

    Type
    Blog Post
    ...suggested roles in transcriptional repression, activity dependent de-repression, chromatin remodeling, gene ...Programmable RNA Editing. Cell Reports 32:107878 . https://doi.org/10.1016/j.celrep.2020.107878 Sinnett SE, ...focused on therapeutic strategies that use gene replacement therapy, gene editing, MECP2 reactivation, RNA...RNA editing, RNA trans-splicing, and protein replacement to reverse the impacts of this disease.  As a...is a strong translational candidate for gene replacement therapy. In 2014, RSRT brought together a strong...have shown exciting and promising results for repairing mutations in models of neurological disease. Klaus... wild-type human iPSCs and then to successfully repair the mutation (Le et al., 2019). Beam Therapeutics...

  12. Fluorescent Proteins 101: Luciferase

    Type
    Blog Post
    ...Fluorecent Proteins in Nano Lanterns Reporter gene assays A reporter gene, such as luciferase, usually serves... is also the most commonly used bioluminescent reporter. This beetle emits a yellow-green light with a...several studies utilized luciferase as a genetic reporter in plant and mammalian cells. Luciferase assays...available to choose from) is now a common feature in reporter plasmids.  Learn How Luciferase Can Be Used In...transcription within cells, where detection of the reporter protein or its enzymatic activity is measured....expression can be determined by detection of the reporter in a specific assay, which ideally would have .... Production of photons by this bioluminescent reporter occurs slower than fluorescent-based methods, ...

  13. Hot Biosensors 2022: Year-End Roundup

    Type
    Blog Post
    ...have newly-available AAV preparations added this year. We’ve added new viral preps of the dopamine sensor...2022.   Find iGluSnFR3 plasmids here! Find viral prep here: pAAV.hSyn.FLEX.iGluSnFR3.v857.PDGFR.codonopt... sustained deep-tissue imaging in vivo. Image reproduced from Liu et al. 2022, under CC-BY-NC license....license.   Find JEDI-2P plasmids here! Find viral preps here: pAAV-EF1a-DIO-JEDI-2P-WPRE and pAAV-EF1a-DIO-JEDI...activity of specific RTKs are limited. In a new preprint, Jared Toettcher and colleagues describe pYtags...of interest and co-expressing a cytosolic ZtSH2 reporter, they observe fluorescence localizes to the plasma...And orthogonal sensors with different ITAM/SH2 reporter pairs allow multiplexed measurement of different...

  14. 3 Tips to Improve HDR Efficiency for CRISPR Editing in Human Cells

    Type
    Blog Post
    ...must undergo homology-directed repair (HDR), a distinct cellular DNA repair pathway. Accomplishing this ...biallelic modifications. Most commonly, DSBs are repaired by the nonhomologous end-joining (NHEJ) pathway... convenient for generating gene knockouts, NHEJ repair does not allow introduction of specific sequence...involves simultaneously introducing a homologous DNA repair template, such as a sin­gle-stranded oligodeoxynucleotide... is relatively rare and DSBs are predominantly repaired by NHEJ. While other groups have focused on strategies...the same allele, probably due to concomitant NHEJ repair. At the PSEN1 locus, over 90% of the HDR edited...one Step 2 – Edit the cells a second time using a repair template that corrects the blocking mutation and...

  15. CRISPR 101: Cytosine and Adenine Base Editors

    Type
    Blog Post
    ... and DNA repair. Black arrows indicate natural cellular processes. BER = base excision repair. Note: hypoxanthine...subsequently converted to thymine through DNA replication or repair. Fusing an inhibitor of uracil DNA glycosylase...Homology-Directed Repair Learn about base editing for plants Read about four base editor reporters Additional...the low editing efficiency of homology-directed repair (HDR), the pathway for resolving DNA double-strand...glycosylase (UGI) to dCas9 prevents base excision repair, which would change the U back to a C base. To increase...newly synthesized” to the cell. Thus, the cell repairs the DNA using the U-containing strand as a template...uracil N-glycosylase (UNG) during base excision repair. Adding a second copy of the UNG inhibitor, UGI...

  16. Starter guide to induced pluripotent stem cells (iPSCs) part 1:  A renaissance in regenerative medicine

    Type
    Blog Post
    ...[1]. In many tissues, stem cells serve to replenish/replace damaged cells that no longer function adequately...stem cells are: Their ability to self-renew or replicate while maintaining the undifferentiated “stem” ... location, mostly to maintain cell numbers or replenish dying cells. For example, hematopoietic stem cells...cells iPSCs are stem cells that are obtained by reprogramming differentiated adult cells by the ectopic expression...in 2006 in Shinya Yamanaka’s lab, where the reprogramming factors were delivered via retroviral infection...via iPSC formation, commonly referred to as reprogramming and, direct conversion from one somatic cell...: Implications for Regenerative Medicine and Reproductive Medicine. Geburtshilfe Frauenheilkd, 2015. 75...

  17. pSiM24: Simplifying Plant Genetic Engineering

    Type
    Blog Post
    ...vectors are cloned and prepped in E. coli. However, they also need to be able to replicate in Agrobacterium ...plant infection, as well as plasmid yield during preparation in E. coli. Addgene depositor Indu Maiti has ...Agrobacterium are termed binary vectors because they must replicate in two species: Agrobacterium and E. coli. Like... Thus, binary vectors contain two origins of replication, one each for E. coli and Agrobacterium, along... the plant, similar to viral vectors’ terminal repeats. It’s a lot of information to fit into a single...bases each. By switching the E. coli origin of replication and codon-optimizing the Agrobacterium ORI, they...efficiency in both types of bacteria. They also replaced the promoter regulating the gene of interest with...

  18. CRISPR-based Genome Editing Systems For Editing in E. coli

    Type
    Blog Post
    ...targeting the pMB1 replicon on pTargetF (Jiang et al., 2015). In addition to the pMB1 replicon, pTargetF contains...In pCas9, we 1) replaced the promoter of gRNA-pMB1 with PrhaB, 2) changed the replicon of pCas into a ... the S. pyogenes Cas9, a temperature-sensitive replicon for self-curing, an arabinose inducible lambda-red... expression of the sgRNA that targets the pMB1 replicon, resulting in cutting of the plasmid. At this ... the gRNA on pCas that is specific to the pMB1 replicon of pTargetF may exhibit a more serious leaky expression...a non-temperature-sensitive replicon, and 3) added the sacB gene for counterselection to yield pEcCas....pEcCas. In pTargetF, we replaced the original 20 bp specific sequences on pTargetF with the ccdB gene containing...

  19. Save Time with Transient Plant Leaf Transformations

    Type
    Blog Post
    ...process by replacing the genes on the T-DNA region with our gene of interest or a reporter gene so that... transformation. Choose good reporter genes There are many great reporter genes available and which one... labor intensive, inefficient, and often not reproducible. Prior to creating transgenic cultures or plants...different treatments on transformation success. Some reporter gene examples are below. The GUS gene (β-glucuronidase...histochemical GUS-staining is a very useful visual reporter (Figure 2). The GUS enzyme cleaves a substrate...transformation success. Luciferases are great quantitative reporters and use a substrate to produce bioluminescence...half-lives then GUS and are therefore good temporal reporters to measure promoter activity, for example. Useful...

  20. The Power Behind NGS Plasmid Validation: seqWell

    Type
    Blog Post
    ... other before proceeding to library prep. Even with this “pre-prep” normalization, it is also often the...many labs. However, the high costs of sample preparation for high-throughput next generation (NGS) sequencing...enhancing the efficiency and simplicity of library prep. As part of our mission, we’ve been working with...Addgene to develop and apply our plexWell™ Library Preparation Technology for NGS-based sequencing and confirmation...Traditional NGS workflows are limited by costly library prep, as each sample requires an individual library. ...plexWell™ workflow is a sequential “pooled library prep”, in which a relatively large number of samples ...understated problem in multiplex NGS library preparation. The challenge here is that, as more samples ...
Showing: 281 - 300 of 956 results