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We narrowed to 957 results for: Cre

Showing: 141 - 160 of 957 results

  1. Beyond the Blood Brain Barrier: PHP.B Enters the Central Nervous System Via the LY6A Receptor

    Type
    Blog Post
    ...PMCID: PMC5529245. Deverman, Benjamin E., et al. "Cre-dependent selection yields AAV variants for widespread...determined LY6A was a receptor used by PHP.B viruses. Creating PHP.B, a neurotropic AAV9 variant PHP.B’s specificity...the Gradinaru labs engineered PHP.B. PHP.B was created by first generating a diverse library of AAV9 mutants... the AAV9 capsid. This mutant library was then screened in vivo in C57BL/6J mice strains to identify variants...injection. Compared to AAV9, PHP.B has a >40-fold increase in CNS transduction and transduces the majority...Ly6a in primary C57BL/6J brain endothelial cells decreased transduction rates while overexpression of the...

  2. New CRISPR Tools: Cas7-11 and PASTE

    Type
    Blog Post
    ... approach proved a challenge. The old standby of CRE/flp didn’t work – it turns out these tyrosine recombinases...likely do it using Prime editing alongside some creative (and significant!) protein engineering. They combined...insert being “dropped” in the wrong place, while increasing the number of potential applications. But they...

  3. Hot Plasmids: Winter 2025

    Type
    Blog Post
    ... The authors have deposited AAV plasmids for Cre-dependent and -independent versions that are trafficked...O’Neill The Lin Lab and collaborators recently created ASAP5, a faster and more responsive genetically-encoded...for its improved kinetics and responsivity in screening ASAP3 variants. B) Electrical and optical recordings...

  4. Adeno-associated Viruses (AAVs) for Genome Editing

    Type
    Blog Post
    ...floxed neoR gene is removed via infection with adeno-cre. AAV-based gene editing compared to CRISPR editing... repair template. It is this application - the creation of sequence-specific knockins - for which the ...Asuri et al, for example, used zinc fingers to increase the efficiency of AAV mediated genome editing ... and encourage to deposit any new AAV tools you create. Many thanks to our guest blogger, Todd Waldman...

  5. Four Base Editing Reporters to Monitor and Enrich Editing in Real-time

    Type
    Blog Post
    ...antibiotic resistance markers, and other enzymes such as Cre-recombinase drastically expanding the base-editing...Base editors create specific point mutations in the genome, but they’re inefficient compared to CRISPR...experiments. In the past few years, scientists have created an array of base editing reporters that can help...Cas9-mediated editing in real time The Harris lab created ACE, a reporter that monitors APOBEC (a family ...and a downstream constitutively active eGFP. To create the inactive mCherry, the lab introduced a 43 base-pair...DSB in their previous reporter, the Harris Lab created a panel of eGFP reporters to quantify the on-target...complexes in real-time (St. Martin et al., 2019). To create the eGFP reporters the Harris lab individually ...

  6. Protocol - pLKO.1 – TRC Cloning Vector

    Type
    Protocol
    ...transforming into bacteria D. Screening for Inserts D.1 Recommended materials D.2 Screening for inserts E. Producing...greater yield of DNA. Back to Top D. Screening for Inserts You may screen for plasmids that were successfully...prevailing guidelines when the pLKO.1 vector was created. Additional design rules can be found at the Broad...prevailing guidelines when the pLKO.1 vector was created. For alternative options, please refer to the enzyme...long-wavelength UV light. Short wavelength UV light will increase the chance of damaging the DNA. Purify the DNA...R0101S NcoI NEB #R0193S Agarose Sigma #A9539 D.2 Screening for Inserts Day 1: 1. Inoculate 5 colonies from...that gives you the optimal viral production. d. Create a master mix of FuGENE® 6 transfection reagent ...

  7. Gibson Assembly Protocol

    Type
    Protocol
    ...about what DNA segments you will need to join to create your final plasmid. Adjacent segments should have...the figures). These identical sequences can be created via PCR with primers that contain a 5′ end that...split an antibiotic resistance gene to effectively create an extra part, one part has half of the antibiotic...unique function for the reaction: T5 Exonuclease - creates single-strand DNA 3’ overhangs by chewing back ...complementary DNA fragments, removing any nicks and creating a contiguous DNA fragment. Pro-Tip Add Extreme...make your own. Transform bacteria with the DNA and screen for the correct plasmid product by restriction ...reaction. However, some labs have observed a sharp decrease in success rate when assembling more than five...

  8. Using a Light Microscope Protocol

    Type
    Protocol
    ...differences create contrast , which allows you to distinguish objects within the sample. You can increase contrast...resolution (the ability to distinguish between two discrete objects). Figure 1 depicts an image of a compound...Diagram of a compound light microscope with labels. Created with BioRender.com. Base Light Source Condenser...objective is in position over your sample. As you increase magnification, you will notice that the objectives...will likely need to re-adjust lighting and focus. Increase the power of the light source by opening the diaphragm...to bring the sample into focus. Pro-Tip As you increase your magnification, remember that the objectives...

  9. Ligation Independent Cloning

    Type
    Protocol
    ...polymerase/exonuclease functions, T4 DNA polymerase can create overhangs of varying length (typically 10-12 bp...distance from their recognition sequence. This can create multiple distinct overhangs with a single enzyme...concentrations in subsequent reactions. Step 3: Create Vector Overhangs Treat the linearized vector with...may be accomplished by gel purification . Step 5: Create Insert Overhangs Treat your purified PCR product...first base removed by the restriction digest to create the free 5' end of the cut vector (shown in blue...across multiple concentrations of insert, thereby increasing your chances of success. Set up a vector only...

  10. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...single copy of DNA (the template), a researcher can create thousands of identical copies using a simple set...oligo primers. This molecular biology technique creates several micrograms of target DNA from just a few...: If you are doing multiple PCRs, save time by creating a “master mix,” which minimizes the number of ...FAQ Tips If your target DNA sequence is GC-rich, increase the time of the denaturing. Your 5’ and 3’ primers... If you are getting non-specific PCR products, increase the annealing temperature step-wise by 1-2°C. ...DNA mutagenesis. A higher cation concentration increases the error rate of the DNA polymerase. Deoxynucleoside...

  11. Protocol - How to Inoculate a Bacterial Culture

    Type
    Protocol
    ...help to increase the density of the culture. Normally cultures shake at 150 - 250 rpm, increase this to...Streaking and Isolating Bacteria on an LB Agar Plate Creating Bacterial Glycerol Stocks for Long-term Storage...Autoclave and allow to cool to room temperature. Now screw on the top of the bottle and store the LB at room... storage of the bacteria, you can proceed with Creating a Glycerol Stock . You can now isolate your plasmid...

  12. Video Library

    Type
    Protocol
    ...collection of Addgene's video content, including how-to screen captures, lab procedure protocols, and career advice...standard laboratory procedures. How-To Videos Screencapture walk-throughs to help guide you through Addgene's... Description Related Page Making LB Agar Plates Create plates to culture bacteria in the lab Pouring LB...with CRISPR Protocol Over-Agar Antibiotic Plating Create plates to culture bacteria in the lab Over-Agar...tips and tricks to reduce transformation time and increase efficiency Bacterial Transformation Protocol Inoculating...

  13. CRISPR Guide

    Type
    Collection
    ...used for large scale edits. These proteins, like Cre recombinase or phage derived serine integrases, insert... GAT; increase nuclease fidelity SpCas9-NG - NG; increase in vitro activity SpG - NGN; increase nuclease...others work to increase Cas9’s proofreading capabilities. No matter the method, increased fidelity enzymes...backbone HypaCas9 - increase Cas9 proofreading and discrimination evoCas9 - decrease off-target effects...domains to expand PAM recognition, increase specificity, and decrease off-target effects Sniper-Cas9 - ...compatible with truncated gRNAs to increase specificity SuperFi-Cas9 - increases fidelity with reduced nuclease...integration of donor DNA. Genome-Wide Screens Using CRISPR In a genetic screen, a population of cells with mutations...

  14. CRISPR Library Amplification

    Type
    Protocol
    ...dramatically increases the chances of individual plasmids being lost from the pool and/or increasing the skewness...plasmids in a single sample. They are often used for screening, barcoding, or other high throughput multiplexed...pipettes Ice slurry (Ice bucket with ice and water to create slurry) 100 mL LB At Addgene we use premixed LB...Immediately purify them! Commercial Maxipreps rely on incremental, ordered cell lysis. E. coli cells are subject...spreading less bacteria on a given plate (example: by increasing the total number of bioassay plates used). Scaling...

  15. Isolating a Monoclonal Cell Population by Limiting Dilution

    Type
    Protocol
    ...well in sparse/individual cultures due to lack of secreted growth factors, so not every cell type will be...cell growth, such as using culture medium with increased serum concentrations, may be optimal for different...monoclonal lines have been sufficiently expanded, screen the lines for transgene expression and/or other...phenotypes. For example, perform Western blotting to screen for lines with the highest or lowest transgene ... vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nature Methods...

  16. AAV ddPCR Titration

    Type
    Protocol
    ...concentration of positive droplets decreases by a factor of ~2. To increase the accuracy of the titer, take...the 96-well plate sealer by gently touching the screen. Prepare the Serial Dilution Place a 48-well dilution...the plate sealer is not at temperature, touch the screen on the plate sealer to allow it to get to temperature...therefore the concentration obtained by ddPCR should decrease by a factor of 2 across the dilutions. In the ...

  17. Lentivirus ddPCR Titration

    Type
    Protocol
    ...the 96-well plate sealer by gently touching the screen. Preparation of the Master Mix Place a ddPCR plate...the plate sealer is not at temperature, touch the screen on the plate sealer to allow it to get to temperature... concentration of RRE positive droplets should decrease by a factor of 2 across the dilutions. RPP30 copies...below, the concentration of RRE positive droplets increases by a factor of ~2 as you progress from the higher...stays relatively even across samples (green). To increase the accuracy of the titer, calculate an average...

  18. Plan Your Experiment

    Type
    Collection
    ...mutant allele of a gene ( point mutant )? Increase or decrease expression of a target gene? Once you have...High-fidelity Cas enzymes increase specificity. Dual-nickase approach increases specificity but is less ...efficiency. In fact, cleavage efficiency may increase or decrease depending upon the specific nucleotides ...alone for mammalian cell lines. Activate (CRISPRa) Increase expression of an endogenous gene(s) without permanently...vivo A common choice for conducting genome-wide screens using CRISPR AAV transduction CRISPR elements are...Transient expression of CRISPR components Expression decreases as RNA is degraded within the cell Short window...window of CRISPR component expression may decrease off-target effects Can be used to generate transgenic ...

  19. Immunology Research Plasmids and Resources

    Type
    Collection
    ...Upon activation, NK cells secrete perforin and granzyme B, two enzymes that create pores within an infected...LSIGN, MGC129964, MGC47866 CREB1 cAMP responsive element binding protein 1 CREB, MGC9284 CTSB cathepsin ...chromogranin A (parathyroid secretory protein 1) CGA CHGB chromogranin B (secretogranin 1) SCG1 CKLF chemokine-like... SDS, SWDS SCG2 secretogranin II (chromogranin C) CHGC, SN, SgII SCGB3A1 secretoglobin, family 3A, member...HIN1, LU105, MGC87867, PnSP-2, UGRP2 SCT secretin - SCTR secretin receptor SR SCYE1 small inducible cytokine... B cells mediate humoral immunity through the secretion of antibodies, and also function as antigen presenting...

  20. Genomic Deletions in Mammalian Cell Lines

    Type
    Collection
    ... Bauer's labs details the use of CRISPR/Cas9 to create genomic deletions in mammalian cell lines. Along... pair of chimeric single guide RNAs (sgRNAs) to create two double strand breaks (DSBs) at a locus in order...strategies for genes and non-coding elements. For creating a gene knockout, two sgRNA located within exons...be 24-mer oligos ( Table 4 ). Design Deletion Screening Primers Design one set of primers internal to ... for future plating. Plate the other half for screening and primer validation (see step 6 ). NOTE: This... allows for sufficient cell proliferation for screening genomic DNA (gDNA) for the intended deletion by...visible with ~2 mm diameter. Primer Validation and Screening for CRISPR/Cas9-Mediated Deletion Isolate gDNA...
Showing: 141 - 160 of 957 results