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We narrowed to 957 results for: Cre

Showing: 261 - 280 of 957 results

  1. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...loxP-stop-loxP cassette into the Rosa26 locus, allowing for Cre-dependent expression of a GMAP inserted gene. The...of a good screening experiment can be found on our blog post entitled “Genome-wide Screening Using CRISPR...Receptor-ome Expression and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library...and later create stable cell lines expressing it. The Church lab fixed this issue by creating a new dCas9...  CRISPR pooled libraries enable researchers to screen the entire genome for genes that regulate a wide...CRISPR knockout library and used this library to screen for genes involved in West Nile Virus (WNV) induced... gRNAs that confer protection to WNV. From this screen the authors were able to identify 7 previously ...

  2. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    ...Wet transfer Increase transfer time Increase voltage Increase SDS/decrease alcohol in transfer...Semi-dry transfer Decrease voltage Decrease transfer time Decrease SDS/increase alcohol in transfer...over-transferring, increase the amount of alcohol and decrease the amount of SDS, with a decreased transfer time...under-transferring, try increasing the amount of SDS while decreasing the amount of alcohol. The increased SDS will ... density of the bands, which increase as protein concentration increases, show the protein concentrations..., you’ll see the signal increase as the concentration of the protein increases (Figure 3A).  This step...linear range (i.e., expression decreases as protein concentration decreases).     Figure 3: Testing...

  3. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ...upon these findings by screening for additional activation domains that increase the expression of target...Synchronization of secretory protein traffic in mammalian cells The journey of secretory proteins, from their... mutagenized and screened for speed and voltage sensitivity. This hierarchical screen led to the generation...has modified the RNA-guided CRISPR-Cas9 system to create a tool that is quick, versatile, and robust. Dubbed...adopt basic biological concepts and employ them in creative and innovative ways. The principle of protein ...molecule imaging and inducible gene expression by creating the SunTag system. The SunTag system, named after...cells without affecting protein function, thereby creating a single-molecule reporter of intracellular processes...

  4. Technical Design of a Western Blot

    Type
    Blog Post
    ...Semi-dry transfer Decreased efficiency Electroblot transfer Increased cost; decreased efficiency ...transfer Increased time Handcast gel Increased time; have to make as-needed Increase specificity...film imaging Increased time; increased consumables cost Decrease background Direct detection method... Wet transfer Increased time Specialty chemical-based buffers Increased cost X-ray film...low-fluorescence PVDF membranes Increased fragility (nitrocellulose) or increased cost (specialty PVDF) ...expressed genes Not as accurate; increased antibody costs; increased optimization Normalizing to total... total protein loading Increased time; increased reagents Good luck and happy blotting! Many thanks...

  5. CRISPR 101: Cytosine and Adenine Base Editors

    Type
    Blog Post
    ...uracil DNA glycosylase. Created with BioRender.com. The BE3 system increased editing frequency to above...editing efficiency decreasing off-target effects In 2016, Akihiko Kondo’s lab created the Target-AID base... offers a 2.3 fold decrease in C→G and C→A products as well as a 2.3 fold decrease in indel formation....nucleobase component of the nucleoside inosine. Created with BioRender.com. How does base editing work...editing Cytosine base editing The beginnings Komor created the first cytosine base editor by coupling a cytidine... which would change the U back to a C base. To increase base editing efficiency, Komor and team wanted...cytidine deaminase portion of the base editor to create SpCas9 base editors with editing windows as small...

  6. New Optimized Genome-wide CRISPRko, CRISPRi, and CRISPRa Libraries

    Type
    Blog Post
    ...contain many sgRNAs per gene to increase confidence in the genetic screen. However, there are benefits to...to conduct genome-wide gain of function screens. These screens can help reveal the function of lowly expressed...large-scale functional screens Read this blog post about genome-wide screening using CRISPR Resources...allowed scientists to easily perform genome-wide screens to effectively and efficiently investigate gene...resources and are particularly useful when trying to screen a limited number of cells like primary cells or... they conducted genome-wide negative selection screens with both A375 (melanoma) and AT29 (colon cancer...metrics to assess library performance in viability screens. Based on these metrics the Brunello library was...

  7. Hot Plasmids - February 2022

    Type
    Blog Post
    ...expanded the genetic screening toolkit by optimizing a Cas12a-based method for screens. Cas12a’s ability ...underutilized in genetic screens because of its lowered editing rate. This research aimed to increase the potential...parallel. Lastly, this Cas12a-based screening method was used to create a genome-wide library capable of ...single cassette provides increased simplicity for combinatorial genetic screens, and this optimized toolkit...imaging of cellular targets Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...10.1038/s41467-019-10846-1.   Optimized genetic screening technique using Cas12a for multiplexed gene perturbation...Gabrielle Clouse While many CRISPR-based genetic screening methods have primarily used Cas9 to knock out ...

  8. CRISPR Kinome Libraries Available: Pooled and Individual Plasmid Formats

    Type
    Blog Post
    ...Library Screening Why the kinome? While properly designed perturbations – in the case of screens using ...at the Broad Institute, John Doench. A genetic screening project can be a tremendous undertaking, producing...technology, gRNAs to knockout genes – are essential screening components, the model system and the assay are...is to do it huge, there are times when a more incremental approach is prudent. What's in the CRISPR kinome... in two sets, each with 4 gRNAs per kinase, so screens can be performed with either 4 or 8 gRNAs per gene...found in genome-wide libraries, and thus can be screened in pooled format at a more-typical scale of tissue.... The individual plasmids will be useful when screening for phenotypes, such as cell-non-autonomous effects...

  9. Enhancing CRISPR Targeting Specificity with eSpCas9, SpCas9-HF1, & HypaCas9

    Type
    Blog Post
    ... target strand and the gRNA. Created with BioRender.com. To decrease the electropositivity of the ...unwanted changes at non-target sites. Means to decrease these so-called “off-target effects” have included...lower on-target efficiency, and sometimes even increase off-target effects, respectively. Recognizing ...School, and the Doudna Lab at Berkeley set out to decrease the off target effects of CRISPR/Cas9 by altering...al. hypothesized that Cas9 cutting efficiency increases when target DNA strand separation is stabilized...higher propensity to reform double helices and decrease Cas9 cutting efficiency. However, this is not ...being cut. Slaymaker et al reasoned that, if they decreased the positive charge in the HNH/RuvC groove, then...

  10. CRISPR 101: Validating Your Genome Edit

    Type
    Blog Post
    ...are ready to begin the screening process. Want to set yourself up for a screening success? Before you start...need to screen hundreds of clones looking for your edit of interest. For all of the screening strategies...phase of your experiment for a screening strategy. Restriction enzyme screening is sensitive to single base...needle in a haystack before going on to generate and screen individual clones. If a clonal line isn’t your ... isn’t a multiple of three will do. A popular screening method for knockouts is Tracking of Indels by ... the minimum number of clones you would have to screen to identify a knockout clone. For example, if your...interest. Thus, to find a null clone, you should screen, at the very least, 4 clones. Did you use a dual...

  11. Corralling and Cornering the Goldilocks Antibody

    Type
    Blog Post
    ...specific markers. The Screening Criteria While choosing which antibodies to screen, we kept in mind our...We therefore use both rAb and mAB in our screening. To screen the antibodies, we have developed a criterion...and his lab members (like me!) are dedicated to creating a public and accessible database for low-cost,...Initiative Cell Census Network, the Wu lab will be screening over 3,000 antibodies for use in mice and human...batch-to-batch variability found in polyclonal antibodies, creating a potentially unlimited supply of identical antibodies...antibodies; Addgene collaborates with these companies to create and provide general availability of their recombinant...to see the reproducibility and accuracy while screening. We also compare different clones of the same ...

  12. xCas9: Engineering a CRISPR Variant with PAM Flexibility

    Type
    Blog Post
    ...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... PAM sequences and/or broader PAM flexibility. Creating xCas9 through directed evolution Hu et al. used...

  13. CRISPR 101: Cytosine Transversion Editors

    Type
    Blog Post
    ...nucleobase component of the nucleoside inosine. Created with BioRender.com. Although it was a huge ...it finds a PAM, Cas9 unwinds the adjacent DNA to create a region of separate strands called an R-loop (...of the enzyme. Figure 2: CRISPR R-loop. Created with BioRender.com. Base editors take advantage...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...GBE, for its reliance on the glycosylase UNG to crease abasic sites (Zhao et al., 2021). Their GBE produced...

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

    Type
    Blog Post
    ...ones? Here, I’ll cover some screening methods to identify them. Visual screening Regardless of the RNAi-trigger...endogenous miRNA precursors can be engineered to created artificial miRNAs. The processed miRNA or siRNAs...conclusion was based upon numerous phenotype discrepancies between gene silencing and knockout methods ...RNAi in the context of a high throughput survival screen found that either technology was equivalently precise... can be divided into three fundamental steps: creation of the RNAi trigger delivering the RNAi trigger...the first stage of an RNAi-based investigation. Creation of the RNAi trigger requires a few initial considerations...these constructs include pGAPi, which allows easy creation of long hpRNA with Gateway-based cloning and straightforward...

  15. Targeted Mutagenesis with EvolvR

    Type
    Blog Post
    ...modular nature, the Dueber Lab created a few versions of EvolvR. To increase the mutation rate of EvolvR,...bacteriophage T7 DNA polymerase increased the processivity of PolI3M, and thereby increasing the editing window of...forward genetics harnesses this at a large scale by screening diverse libraries of genetic variants. Several...nucleotides. Both the Bassik Lab and the Chang Lab have created methods that introduce diversification by targeting... integration of oligonucleotide libraries of a discrete size at the target site. The use of such libraries...occasionally introducing mutations (red nucleotide). Created with BioRender.com. The initial version of ...per generation of wild-type E. coli, while only increasing the standard mutation rate seen during DNA replication...

  16. Supporting Rare Disease Research with the 2020 BeHEARD Awards

    Type
    Blog Post
    ... Association for Creatine Deficiencies: Compensating for SLC6A8 mutations in Creatine Transporter Deficiency...SLC6A8 gene, also known as the creatine transporter. The Association for Creatine Deficiencies (ACD) is a parent-let...regeneration of ATP from ADP is creatine. In order to reach the brain, creatine needs to be transported from...Deficiency Creatine Transporter Deficiency (CTD) is a rare X-linked inherited neurometabolic condition and... and one of three Cerebral Creatine Deficiency Syndromes (CCDS) that present as global developmental delays... blood and cross the blood brain barrier using creatine transporters. However, mutations in the transporter...SLC6A8 impede this transport and result in global creatine deficiency, which is characterized by development...

  17. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    ...libraries here at Addgene, including cDNA, barcoding, screening, and gRNA libraries. Most of the pooled libraries...determined, larger lentiviral preps can be used for screening-scale transduction of cells, resulting in cell...library. These cells can then be used for genetic screening, followed by sequencing and further data analysis...some of your plasmids didn’t amplify after your screen is completed!  Producing Your Lentiviral Library... any issues.  Keep in mind that a low MOI will increase the chances of a cell being infected by only one... one viral particle, but it also increases the chances that a cell doesn’t get infected at all. To make...as seeding density and reagent concentration.  Screening time Phew! You are now (finally!) ready to use...

  18. Hot Plasmids - December 2019 - CRISPR pooled library, LipoGlo, CRISPRa, and Voltage Imaging Tools

    Type
    Blog Post
    ...pooled libraries have been created in lentiviral systems for mammalian screens, which enables researchers... as the screen readout. This retroviral mouse CRISPR pooled library can be used for screening of cells...pooled libraries are popular tools for genetic screens, enabling genome-wide interrogation of the genes...required for cellular processes. For a successful screen, it is necessary to identify the gRNAs that elicit...are poorly transduced by lentivirus, and thus screening with available CRISPR libraries was not possible...solve this issue, Sarah Teichmann and colleagues created a retroviral CRISPR knockout library using gRNAs... from mice that constitutively express Cas9 and screen for genes that affect Th2 differentiation and activation...

  19. How to Negotiate a Successful Lab Start Up Budget

    Type
    Blog Post
    ...goals and the resources. Instead of creating a start up budget, create a start up list.  In this post we...award by giving you step by step instructions on creating a budget, making decisions based on that budget...budget, and negotiating in regards to the budget. Creating a budget We start by using your research specific...budgetary needs from your experimental goals. When creating your budget, you should take the following steps... that timeline Avoid writing down prices as you create your list. This helps to ensure that all of the...”, “colony”, etc. This will help save time by screening out irrelevant candidates, and, more importantly...doesn’t have to be complicated, or difficult to create this list. Start BEFORE searching for a faculty...

  20. Stabilized Bacterial Promoters: Constant Gene Expression at any Copy Number

    Type
    Blog Post
    ...gene expression. By itself, increasing the copy number of a gene increases its expression. However, if...as plasmid copy number increases, the amount of repressor protein also increases thereby intensifying repression...When they increased concentrations of IPTG, a constitutive promoter upstream of GFP increased expression...that is responsive to a repressor protein, the increase in expression due to copy number is kept in check...origins of replication, the stabilized promoters created by Segall-Shapiro et al. showed nearly no position-dependent...stationary phase, the number of plasmids per cell increases 4-5 fold (for pUC plasmids) or ~2 fold (for p15a...compared to a constitutive promoter. To simulate the increase in plasmid copy number during the transition to...
Showing: 261 - 280 of 957 results