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We narrowed to 736 results for: RAN-1

Showing: 101 - 120 of 736 results

  1. DNA Purification Without a Kit

    Type
    Blog Post
    ...just make their own buffers when the kits buffers ran out. Homemade buffers performed the same as those...series to increase binding capacity. Figure 1: Purify DNA using glass filters. Image: Kim and Morrison...a gravity-based column plasmid maxiprep kit takes 1-1.5 hours to complete, while this syringe-based method... solution. NaI has two purposes in this protocol: 1) it solubilizes both DNA and agarose, and 2) it helps...property and developed a protocol that uses Whatman No. 1 paper or even paper towels to purify DNA and RNA from...rather a glass powder slurry that has a milk-like appearance. To make DNA stick to the glass particles in ...the Patterson Protocol was shown to purify DNA ranging in size from 100 bp to 48 kb with high yields and...

  2. Great Results Start with Great Standard Curves

    Type
    Blog Post
    ...    Figure 1: A 1 mg/mL control is diluted in a series ranging from 1:2 to 1:32. Each individual...series that ranges from 1:2–1:32. The specific dilution series used will depend on the expected concentration...the data points fit the trendline. R2 ranges from 0 to 1 with 1 being a perfect fit and 0 indicating that...example, Figure 1 depicts a 2-fold dilution series. Each step of the series dilutes by 1:2 for a series...individual step of the series is consistent at 1:2. When preparing a dilution series, use a new pipette tip for...concentration lies within the dynamic range of the curve. The dynamic range is the linear span between the lowest...the dynamic range of your standard curve, so if your sample does not fall within this range, it may be ...

  3. Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)

    Type
    Blog Post
    ...cells transduced with lentiCas9-blast. A549 cells were transduced (MOI = 37) and selected with 1 µg/mL...expressing Cas9 via transduction with Cas9 lentiviral vector, lentiCas9-Blast (52962-LV, Figure 1). Some cells...were frozen down. Figure 1: Cas9 expression in A549 cells transduced with lentiCas9-blast. Anti-Cas9...of the gel in the BRAF gRNA-transduced samples (data not shown). Pro-Tips: 1.Designing primers against ...The entire process from start to finish took about 1 month. Cas9 delivery Overview: Before any editing...Western blotting is a loading control. Pro-Tips: 1. It’s all about the MOI. In retrospect I used a way... pool had wildly variable Cas9 expression (Figure 1). This is a problem because Cas9 expression affects...

  4. Viral Vectors 101: AAV Variables That Matter

    Type
    Blog Post
    ...). Analysis of Transduction Efficiency, Tropism and Axonal Transport of AAV Serotypes 1, 2, 5, 6, 8 and...Overview. Cells, 12(5), 1–41. https://doi.org/10.3390/cells12050785 Murlidharan, G., Samulski, R. J., &... ONE, 9(8), 1–10. https://doi.org/10.1371/journal.pone.0106472 de Backer, M. W. A., Brans, M. A. D., Luijendijk...vector-mediated transduction of the corticospinal tract: Comparison of four promoters. Gene Therapy, 28(1–2), 56...the selection process. Factors to think about are (1) your experimental conditions and measurements; (2...serotype, promoter, and titer.     Figure 1: Flow chart depicting a) steps for selecting serotypes...injection site than AAV2 (Watakabe et al., 2015) (Figure 1). You’ll need to consider if such properties could...

  5. AAVs CREATed for Gene Delivery to the CNS and PNS

    Type
    Blog Post
    ... Table 1: Transduction efficiency of AAV-PHP.B versus AAV-PHP.eB in the CNS. A GFP transgene was packaged...marker). Check out tables 1 and 2 below for a summary of the quantified transduction efficiency of AAV-PHP.eB... AAV.PHP.S’s great neuronal transduction efficiency, as shown in tables 1 and 2, with a cell-type specific...nervous system (CNS). AAVs are popular tools because: 1) their genomes are easy to manipulate, 2) they have... AAV vectors. What can these AAV’s be used for?  1. Multi-color labeling of neurons Multi-color labeling... dendrites extend to. The keys to this method are 1) high levels of color diversity so that cells are ...expression into a two part system (figure 4d):  Part 1: A cocktail of three AAV vectors expressing RGB proteins...

  6. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    ...outside of the antibody’s dynamic range. Specifically, the 1:2, 1:1, and No dilution samples all have ...dilution range from 1:100–1:1,000. If your antibodies come from ascites fluid, try a dilution range from ...with a recommended dilution and/or range (typically between 1:500 and 1:10,000 for primary antibodies). ...from 1:1,000–1:100,000. Secondary antibodies For secondary antibodies, the recommended dilution range is ...is usually between 1:5,000 and 1:200,000. Check the manufacturer’s recommendations, and if needed, optimize...marked on the membrane (1) using the gel as a guide. The gel is then removed from the membrane (2) and the...Mark your membrane! Membranes look the same from both sides, so it’s quite easy to think lane 1 is‌ lane...

  7. Golden Gate Assembly Upgrades: More Fragments, Faster Assembly, and Higher Fidelity

    Type
    Blog Post
    ... assembly reaction** 1 5 min., 37 ºC (2.5 μl) 687 100% 274,200 2,742,000 1 60 min., 37 ºC (2.5 μl)... volume of the 1 ml outgrowth spread on each transformation plate can be manipulated to result in appropriate...Figure 3, Table 1). These improvements (5- to 12-fold increases in the number of transformants while maintaining...expected complete sequence for the lacI/lacZ genes (1), while sequencing of white colonies showed a mixture... obtained from 1-, 12- and 24-fragment assemblies of the lacI/lacZ cassette, and illustrates how the volume...fidelity studies conducted by Potapov et al. at NEB (1, 2). While T4 DNA Ligase, the mainstay of most biotechnological... 16 ºC) x 30 (100 μl) 78 90.7% 783 9,792 Table 1. Yields and fidelities for Golden Gate Assemblies ...

  8. Plasmids 101: Common Lab E. coli Strains

    Type
    Blog Post
    ...Table 1 below outlines a few of the more common genetic changes found in E. coli strains. Table 1: Common...amber (UAG) stop codon by tyrosine insertion λ-thi-1 or thi1 Mutation in thiamine metabolism Requires exogenous... all based on E. coli K-12 and are considered BSL-1. Table 2: Lab strains of E. coli Strain Natural...plasmids, blue/white screening. F- endA1 glnV44 thi-1 recA1 relA1 gyrA96 deoR nupG Φ80dlacZΔM15 Δ(lacZYA-argF...) recA13 leuB6 ara-14 proA2 lacY1 galK2 xyl-5 mtl-1 rpsL20(SmR) glnV44 λ- JM109   General cloning and...strain for cloning repetitive DNA. endA1 glnV44 thi-1 relA1 gyrA96 recA1 mcrB+ Δ(lac-proAB) e14- [F' traD36...of an E. coli K-12 strain. F- λ- ilvG- rfb-50 rph-1 NEB Stable   For cloning into and storage of lentiviral...

  9. Lambda Red: A Homologous Recombination-based Technique for Genetic Engineering

    Type
    Blog Post
    ...recombineering system has three components (Figure 1): 1) Exo, 2) Beta, and 3) Gam. All three are required...generate 2 possible products: 1) a partially dsDNA duplex with single-stranded 3’ overhangs or 2) if the ...sequence of the translated protein.  These changes can be 5’ or 3’ of the desired change.   Table 1: Overview...recombineering with an ssDNA oligo substrate.   Figure 1: Components of the Lambda Red Recombineering system...and tags.  The typical frequency of recombinants is 1 positive clone out of 104 to 105 colonies. Pro Tip... with a recombination frequency between 0.1% to 1%, and can be increased to as high as 25-50% by designing...replication. There are two ways to avoid activating MMR: 1) use a strain of bacteria that has key MMR proteins...

  10. COVID-19 Resources

    Type
    Collection
    ...infection to host cells . Signal transduction and targeted therapy, 5 (1), 283. https://doi.org/10.1038/...envelope protein, E; nucleocapsid protein, N; and membrane protein, M), including plasmids for protein production...Anderson et al., 2020). BSG plasmids - (CD147), transmembrane glycoprotein of the immunoglobulin superfamily...proteolysis of SARS-CoV Spike protein, activating its membrane fusion function (Bosch et al., 2008). TACE plasmids... protease (Mpro) activity reporters Reverse transcriptase, protease, and polymerase constructs for SARS-CoV...Llamas Required - Synthetic Nanobodies Against Membrane Proteins Return to top Pooled Libraries Pooled...

  11. CRISPR Between the Genes: How to Experiment with Enhancers and Epigenomics

    Type
    Blog Post
    ...They help us to transform raw genomic coordinates into meaningful experiments. Table 1: An emerging family... chromosome, start, and stop. For instance (chr3, 1, 10) indicates a stretch of DNA at the very beginning...genetic elements like protein, RNA, and DNA (Table 1). As with GPS in the real world, coordinates alone...enhancers with the help of genome math. Figure 1: Genome math illustrated. Intersect (left) and set...histones with “GPS for proteins,” ChIP-seq from Table 1. Because of DNA’s 3D geometry and the chemical properties...experiment to identify the critical NANOG binding sites: 1. Find Enhancers that have NANOG binding sites 2. Design... In order to denote the NANOG enhancers from step 1 with genome math, we’ll need a bit of shorthand from...

  12. Antibodies 101: Normalization and Loading Controls for Western Blots

    Type
    Blog Post
    ...like uneven pipetting or irregular membrane transfer. Figure 1: A loading control is a type of positive...because the loading control band is weaker in condition 1 than condition 2, the normalized values for the two...to using loading controls accurately. Assumption #1: The loading control is not perturbed by the experiment...without your knowledge? If you can’t trust Assumption #1, any change in the normalized intensity of your POI...interest band is significantly lighter in “condition 1” than “condition 2.” By eye, the loading control appears... the protein abundance axis, both blue “condition 1” points (POI and loading control) are smaller than...assumptions discussed above into question. Assumption #1: The loading control is not perturbed by the experiment...

  13. Viral Vectors 101: Producing Your rAAV

    Type
    Blog Post
    ...Inactivation of AAV Serotype 1 Vectors. Human Gene Therapy Methods, 28(1), Article 1. https://doi.org/10.1089...commonly called “triple plasmid transfection” (Figure 1).    Figure 1: Overview of key steps of AAV production...repeat sequences. Human Gene Therapy Methods, 23(1), Article 1. https://doi.org/10.1089/hgtb.2011.034 Benyamini...in the CNS. Molecular Neurodegeneration, 15(1), Article 1. https://doi.org/10.1186/s13024-020-00361-z ..., expressing Ad E1A and E1B, can be transfected with your transfer plasmid, carrying your gene of interest... performed 12–18 hours post-transfection to help remove toxic transfection reagents and keep the cells...cells happy. Harvesting virus Once you have transfected your cells, wait 2–5 days and then collect the supernatant...

  14. Opto-Nanobodies: Using Light to Manipulate Cell Signaling and Protein Purification

    Type
    Blog Post
    ...loops 1 and 6 resulted in light controlled binding, but with opposite effects: insertion into loop 1 caused...experiments.  AsLOV2 Inertion Site mCherry #1 mCherry #2 GFP Loop 1 dark-induced dark-induced dark-induced...favorite protein (YFP). You have two options:  Option #1: Affinity tag purification You tag YFP and use an ...and one against GFP. Insertion of AsLOV2 into loop 1 always generated OptoNBs with dark-induced binding... induced dark-induced blue-light induced Table 1: Summary of the effect of AsLOV2 insertion site on...let the team detect its co-localization with a membrane-localized mCherry or GFP protein. With this system...bind, it co-localizes with its target at the cell membrane and when the OptoNB can’t bind, it localizes to...

  15. The PAM Requirement and Expanding CRISPR Beyond SpCas9

    Type
    Blog Post
    ...doi.org/10.1186/s13059-018-1591-1 Cox DBT, Gootenberg JS, Abudayyeh OO, Franklin B, Kellner MJ, Joung J, Zhang... 556:57–63 . https://doi.org/10.1038/nature26155 Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz... 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...SpCas9 variants effectively double the targeting range of CRISPR/Cas9 within the human genome. Another ...known to produce off-target editing with SpCas9 (Amrani et al., 2018). When choosing a Cas9, remember to...

  16. X-CHIME: Context Dependent Germline Knockout in Immune Cells

    Type
    Blog Post
    ...in the immune system. Nature Immunology, 25(1), Article 1. https://doi.org/10.1038/s41590-023-01689-6 ...expression vectors (Table 1). The four systems Name Function Plasmid Transduction Marker Mouse Strain... GFP (vex) Rosa26-FlpO-ERT2; H11-Cas9 Table 1: X-CHIME systems Proof of Concept These systems were...was lethal (LaFleur et al., 2024).   Figure 1: Schematic showing the X-CHIME approach. Figure from...genes in the immune system. Nature Communications, 10(1), 1668. https://doi.org/10.1038/s41467-019-09656-2... LaFleur et al., 2019b). CHIME works through transduction of Cas9-expressing (Rosa26-Cas9) hematopoietic... a lentiviral vector containing a gRNA. These transduced stem cells can be implanted in irradiated recipients...

  17. Who Gives a Tweet? 9 Facts About Scientists on Twitter

    Type
    Blog Post
    ...2017. 1. Among scientists, historians have the most Twitter users. Biologists rank #8. Figure 1: Highly...that these results could be biased for two reasons: 1) they are based on the US Occupational Employment ...female to male = 0.629 (the closer this ratio is to 1, the closer males and females are to parity - the ... authorship in the United States (0.428). 4. The #1 (re)tweeted domain by scientists was Instagram Social...links to research that isn’t behind paywalls. 5. The #1 (re)tweeted science domain by scientists was Nature...Discipline. Biologists, neuroscientists, and ecologists (orange box) post tweets containing scientific domains ...network of life (green), physical (blue) and social (orange)  scientists largely cluster by discipline, suggesting...

  18. The Effect of COVID-19 on Liu Lab Plasmid Requests From Addgene Data

    Type
    Blog Post
    ...was around 33% of that observed between Jan. 1 and Mar. 1, 2020. Figure 2: A simple approximation...paper. Green and red lines depict the sum of mean +1 and -1 std for each paper, respectively. The fitted...complication in the China data – there is a period of 1 month, from 1/22/20–2/23/20, where there were zero plasmid... from 5/22/14 to 5/19/20). Each paper has between 1–32 plasmids and a total of 2–2,590 requests. We received...correspond to publications of popular papers. Figure 1: Plasmid requests by date. Parentheses depict the ...China. However, it is evident that if we believe that 1/22/20–2/23/20 are the more accurate dates of lockdown...cases being reported (right, red), likely due to quarantining {% video_player "embed_player" overrideable...

  19. MXS Chaining

    Type
    Blog Post
    ...assembly. Journal of biological engineering. 2010;4(1):1. Pubmed PMID: 20205762. PubMed Central PMCID: PMC2822740...with minimal spectral overlap between them (Table 1). In each construct, 3 copies of the fluorescent protein... enabled labeling of particular structures (Table 1). Each construct was flanked with a CMV promoter (...construct was then introduced to HeLa cells (Figure 1). The resulting HeLa cells show robust labeling, with...fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave researchers the ability ... all four constructs into one 15 kb insert. Table 1: MXS Constructs for Labeling Cellular Structures ...maxima Tethering partner Subcellular localization 1 TagBFP 399nm/ 456nm histone 2B (H2B) Chromatin 2...

  20. Recombinase-based State Machines Enable Order-dependent Logic in vivo

    Type
    Blog Post
    ...endogenous gene expression (2). Our paper (1) explains how to arrange attB-attP pairs on a register to record...recombinase-based state machines in living cells” (1), into a more straight-forward, concise explanation...an RSM is to sequence the register. In our paper (1), we allowed individual bacterial cells to form colonies...in the Materials and Methods section of our paper (1). Lastly, it is possible to build gene regulatory ... not. How might one implement RSMs? In our study (1), we implement RSMs in E. coli with two plasmids (...performance data from the indicated figures in our paper (1)) Input Plasmid Output Plasmid Fig. 3A pNR64 pNR160...in the Material and methods section of our paper (1). How might one troubleshoot GSRMs that don't work...
Showing: 101 - 120 of 736 results