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We narrowed to 328 results for: hal.1

Showing: 141 - 160 of 328 results

  1. Targeted Mutagenesis with EvolvR

    Type
    Blog Post
    ...activity degrades the displaced strand.   Figure 1: EvolvR consists of an error-prone polymerase (PolI...mutation rate and extend the editing window of EvolvR: (1) two additional mutations to PolI3M generated PolI5M...components of EvolvR and its potential applications. Challenges of existing mutagenesis methods Several continuous...drawbacks with these approaches that makes it challenging for users to achieve targeted diversification...PMID: 30253789. PubMed Central PMCID: PMC6154929. Halperin, Shakked O., et al. "CRISPR-guided DNA polymerases...

  2. Hot Plasmids: Summer 2024

    Type
    Blog Post
    ...10.1038/s41551-024-01227-1. doi:  https://doi.org/10.1038/s41551-024-01227-1   CHARM: A compact epigenetic...spacer proteins ending with a GFP nanobody (Figure 1). This enrichment streamlines the purification and...highly-visible beads on the cryo-EM grid.     Figure 1: MagIC-cryo-EM for sample enrichment and structure...single-particle analysis of PRC2 with RvLEAMshort (1:6 molar ratio) with 10 minutes of glutaraldehyde crosslinking...myriad cell types and subtypes, and a familiar challenge for anyone who has done histology on brain tissue...

  3. Selecting Your Plasmid Purification Kit

    Type
    Blog Post
    ...require large amounts of high quality DNA. Figure 1 illustrates the three grades of DNA you may need based...their relative quality and quantity.   Figure 1: Plasmid preps fall into different categories based...specific volume ranges of culture as their input (Table 1). When you feel like you need just a bit higher DNA...kit for example. These kits are designed to handle ~1–5 mL of liquid culture. Using a higher volume can ...beads and a suitable magnet as you scale up. Table 1: Standard Prep Kit Capacities* Prep size Amount...Amount of culture Approximate DNA yield** Miniprep 1–5 mL 5–50 µg Midiprep 25–100 mL 20–200 µg Maxiprep...inks Reduced cardboard packaging by more than half 70% recycled materials Sustainable forestry ...

  4. Plasmids 101: Degron Tags

    Type
    Blog Post
    ...systems reviewed here. The only required components are 1) a SMASh-tagged protein and 2) asunaprevir (small ... tag size, which can also be handy!   Fig 1. dTag molecules induce dimerization of an FKBP12F36V...Nabet et al., Chemical Biology, 2018.    HaloTag The HaloTag was initially developed as an affinity tag...the Halo-tagged protein in question, it is targeted to the proteasome for degradation. The HaloTag is ...straightforward system – HaloTag and its PROTAC are all that is needed to facilitate degradation. HaloTag is also the...visualization of proteins of interest. A fluorescent Halo ligand can be added to media, buffer, etc., which...proteolysis-targeting chimera (PROTAC) was developed for HaloTag. PROTACs are engineered molecules which target ...

  5. The PAM Requirement and Expanding CRISPR Beyond SpCas9

    Type
    Blog Post
    ... modify any desired genomic target provided that (1) the sequence is unique compared to the rest of the...will cover three ways to circumvent this limitation: 1) the use of novel S. pyogenes Cas9 variants with varying...Cas9 from Staphylococcus aureus (SaCas9) is about 1 kilobase smaller than SpCas9, so it can be packaged...Biol 19: . https://doi.org/10.1186/s13059-018-1591-1 Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, ...WX, Scott DA, Gootenberg JS, Kriz AJ, Zetsche B, Shalem O, Wu X, Makarova KS, Koonin EV, Sharp PA, Zhang...

  7. Plasmids 101: Dimers and Multimers

    Type
    Blog Post
    ...indicates the presence of a multimer.   Fig. 1: The monomer of Plasmid 12259: pMD2.G (depicted on...plasmid sequencing services using long-read sequencing (1-25 kb), such as Oxford Nanopore sequencing or PacBio...10017-26. doi: 10.1074/jbc.M110.176636. Epub 2011 Feb 1. PMID: 21285356; PMCID: PMC3060453. Bazaral M, Helinski...multiple circular DNA forms of colicinogenic factor E-1 from Proteus mirabilis. Biochemistry. 1968 Oct;7(10...Methods Mol Biol. 2009;582:11-25. doi: 10.1007/978-1-60761-340-4_2. PMID: 19763938. Maucksch C, Bohla A... PMID: 32338220. Crozat E, Fournes F, Cornet F, Hallet B, Rousseau P. Resolution of Multimeric Forms of...

  8. Viral Vectors 101: Inverted Terminal Repeats

    Type
    Blog Post
    ...) vector (Figure 1). Everything else outside the ITRs gets left behind.    Figure 1: Recombinant AAV ...productivity but increases transgene expression. Sci Rep, 7(1), 5432. https://doi.org/10.1038/s41598-017-04054-4...10.1089/hgtb.2016.133. PMID: 28967288. Shitik, E. M., Shalik, I. K., & Yudkin, D. V. (2023). AAV- based vector...

  9. Human Germline Editing Using CRISPR

    Type
    Blog Post
    ... employing HDR. Ma et al. moved forward with gRNA 1 to conduct microinjection experiments in S phase zygotes...instead from the mother’s wild type allele (Figure 1). 72.4% of embryos carried two wild-type copies of...proceeded through NHEJ rather than HDR. Figure 1: Homology-directed repair in targeted zygotes proceeds...heterozygous mutations, it will clearly be much more challenging for homozygous mutations. Moreover, introducing...

  10. Tagging Optogenetics and Chemogenetics Receptors: Fluorescent Proteins and Other Options

    Type
    Blog Post
    ...were found on the plasma membrane (top panel in Fig. 1). Of course, this was problematic, because the DREADD...plasma membranes in both species (bottom panel in Fig. 1). Our results indicate that the tag protein may have...protein of interest the membrane.   Figure 1: Electron microscope images showing ultrastructural... M, Mueller SAL, Roseboom PH, Oler JA, Kalin NH, Hall RA, Smith Y (2019) Ultrastructural localization ...

  11. RNA Extraction Without A Kit

    Type
    Blog Post
    ...glassware should be treated with a DEPC solution for 1 hour, and autoclaved to remove residual DEPC. Alternatively...more large, expensive ultracentrifuge.  Figure 1: An outline of the different steps in RNA extraction...extraction: twenty-something years on. Nature Protocols 1:581–585. https://doi.org/10.1038/nprot.2006.83 Additional...until homogenization and cell lysis. This can be challenging depending on your laboratory situation or tissue...

  12. Plasmids for Endogenous Gene Tagging in Human Cells

    Type
    Blog Post
    ...Collection Fluorescent tagging strategy Figure 1: Gene editing is the addition of a useful coding sequence...plasmid design strategy. A donor plasmid contains 1 kb homology arms matching the gene of interest, a ...Institute website at alleninstitute.org. References 1. Roberts, Brock, et al. "Systematic gene tagging using...Institute website at alleninstitute.org. A classic challenge in cell biology is making sure that what we observe...

  13. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
    ...the tag sequence flanked by homology arms (Figure 1). In the first step of optimization, we started off...form the ribonucleoprotein complex (csRNP, Figure 1). We use in-house purified Cas9 protein, which, in...standard immunocytochemistry (ICC) assays.  Figure 1: Three ingredients of epitope-tagging experiments....daydreaming about sunny days in the UK.   References 1. Bressan RB, Dewari PS, Kalantzaki M, Gangoso E, Matjusaitis...Creating epitope-tagged clonal cell lines remains a challenge as it relies on inefficient homology-directed ...representing a protein super family). To overcome these challenges, we set out to develop a hassle-free, highly ...

  14. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

    Type
    Blog Post
    ...sticks out and can be considered a linker (figure 1). In fact, for several FRET biosensors (yellow cameleon...fluorescent protein are relatively close together (figure 1). Where should the fluorescent protein be inserted...fluorescent protein database." Nature methods (2019): 1. PubMed PMID: 30886412. Mastop, Marieke, et al. "A...properties of the protein of interest can be challenging. In this blog I discuss strategies to generate... (catalytic activity or interactions) can be challenging. If a fusion can complement a knock-down or knock-out...

  15. Harnessing Bacterial Toxins for Allelic Exchange

    Type
    Blog Post
    ... requires selection. These two steps are (Figure 1): The initial plasmid integration step (the so-called...antibiotic resistance marker from the vector (abR, Figure 1). The resolution step (“double crossover”) which leads... loss of a specific phenotype (using sacB, Figure 1, further discussed below).  Counter-selectable marker...Current protocols in molecular biology 106.1 (2014): 1-16. PubMed PMID: 24733238. Additional resources on... revolutionized allelic exchange. Even now, only half our lab has switched over to pTOX. However, we feel...

  16. Special Delivery: Fluorophore Targeting for FRET Studies

    Type
    Blog Post
    ...standard for genetically-encoded FRET donors (Fig. 1). Scores of FP variants have been created, thereby...with finesse using tRNA suppression technology (Fig. 1) (Dumas, Lercher, Spicer, & Davis, 2015). By using...orthogonal labeling strategies are presented (Fig. 1) which can be carried out using Addgene plasmids. ...involved in skeletal muscle contraction.   References 1. Degorce, François, et al. "HTRF: a technology tailored... transfer measurements of ryanodine receptor type 1 structure using a novel site-specific labeling method...FRET distance measurements (for an example see Mahalingam et al., 2014).  Many thanks to our guest blogger...

  17. AAV Titration by qPCR Using SYBR Green Technology

    Type
    Protocol
    ...dilution Dilution 1 (DNase step) 5 uL AAV stock 45 uL 10X 10X Dilution 2 5 uL Dil. 1 95 uL 20X 200X Dilution...range from 1 x 10 12 GC/mL to >2 x 10 13 GC/mL and we use an internal reference virus that is 1 x 10 13 ...Equipment qPCR instrument Heating plate Pipettors 1–10 µL single channel pipette 20–200 µL single channel...channel pipette 200–1000 µL single channel pipette 1–10 µL multichannel pipette 2–50 µL multichannel pipette...The reference material should have a titer within 1-log of the expected titer of the samples being tested...Amount for ONE Reaction Amount for 100 reactions (1 x 96 well plate) Universal SYBR Master Mix 2X 10 μL...mole)/bp = 4.03 x 10 6 g/mole Moles/μl: 1.07 μg/μL x 1 g/10 6 μg x1 / 4.03 x 10 6 g/mole = 2.65 x 10 -13 ...

  18. Hot Plasmids - November 2023

    Type
    Blog Post
    ... depending on the specific edit.   Figure 1: Choosing the best PE6 variant for your application...modular envelope design. Nature Communications, 14(1), 5141. https://doi.org/10.1038/s41467-023-40788-8...externally-timed bands of color with HaloTag-iPAK4 by applying different HaloTag dyes and showed they could record...local proteome, but some applications have been challenging or inaccessible. The Ting lab recently made two.... B) HEK cell with an iPAK4 fiber labeled with HaloTag-iPAK4. Scale bar 10 µm. C) Images of iPAK4 fibers...fibers in cultured rat hippocampal neurons. HaloTag dyes are switched from yellow to red at 0 h and back...

  19. Using Ultrasound to Image Bacteria in vivo: Acoustic Reporter Genes

    Type
    Blog Post
    ...optimized for expression in E. coli: acoustic gene 1 (arg1) and acoustic gene 2 (arg2). The key difference... plasmids are available from Addgene! References 1 Bourdeau, R.W., Lee-Gosselin, A., Lakshmanan, A., ...widely available imaging platform: ultrasound. Challenges of in vivo imaging with existing bacterial reporters...

  20. Gibson Assembly Protocol

    Type
    Protocol
    ...ssDNA. (Rabe & Cepko, 2020). Incubate the mix for 1 hour at 50 °C or follow manufacturer's instructions...part, one part has half of the antibiotic gene and the adjacent part has the other half. Any colonies should...
Showing: 141 - 160 of 328 results