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We narrowed to 587 results for: cel.2;

Showing: 501 - 550 of 587 results

  1. Reaching out to China: Canton Nucleic Acids Forum (CNAF) 2015

    Type
    Blog Post
    ...systems to defend against both DNA and RNA viruses - 2 cas systems regulate at the DNA level and 1 at the...origin of cellular life. His lab has long hypothesized that life started with RNA and a simple cell membrane... circular RNA they have identified resulted in a cell migration defect. They have created a circRNA website...

  2. Addgene's Cancer Collection Pages Connect the Research Community

    Type
    Blog Post
    ...: PMC4353587. 2. Stephen, Andrew G., et al. "Dragging ras back in the ring." Cancer cell 25.3 (2014): ...molecular level, at the cellular level there is a universal theme - uncontrolled cellular proliferation. We ...cancerous cells Luckily, there are several safety mechanisms in place to protect us from rogue cells when ...process of programmed cell death, or apoptosis, is one such mechanism. When a cell or its genetic material... directly inhibit the ability of a cell to undergo programmed cell death while simultaneously stimulating...eliminate every cancerous cell. Even so, to pose the greatest threat, cancerous cells still need to successfully...universally-dividing behavior of cancer cells, attacking the cellular machinery necessary for division. This...

  3. Rabies and Neuronal Tracing

    Type
    Blog Post
    ... 2008 Dec; 18(6): 617–623. Pubmed PMID 19349161.  2. Deshpande A, Bergami M, Ghanem A, Conzelmann KK, ...it first infects one cell (the “starter cell”). It then replicates within that cell, and nascent RABV cross...connections to the starter cell, RABV was modified to only infect user-defined starter cell types, as well as ...dictate infection of target cells through their interactions with target cell surface receptors (Callaway...Callaway 2008). Based on this, cell type specificity of the starter cell can be achieved by using EnvA-pseudotyped...studied biological processes, like how our stem cells are housed in specific locations and give rise to...Tracers Due to their ability to efficiently enter cells and deliver a variety of cargoes, genetically engineered...

  4. Your Lentiviral Plasmid FAQs Answered

    Type
    Blog Post
    ... replication competent virus [usually HIV-1]; and 2) the potential for oncogenesis through insertional...of interest into cells. Unlike gamma-retroviruses that can only infect dividing cells, lentiviruses can...dividing and non-diving cells while retroviruses can only infect dividing cells. When choosing the appropriate...to ensure that the proper cell line is used; several common laboratory cell lines including 293 were immortalized...can infect dividing and non-dividing cells.  Addgene has an extensive collection of lentiviral plasmids...transfecting four different plasmids into the producer cells (two packaging plasmids, an envelope plasmid, and...appropriate gene delivery system, the specific target cell or tissue needs to be considered. Q2: Can I use...

  5. Hot Plasmids February 2024

    Type
    Blog Post
    ...Figure 2: Inner mitochondrial membranes imaged with structured illumination microscopy in HeLa cells expressing...yielded editing of 80–90% in human cell lines and primary cells. Although cell entry of a Cas9 ribonucleoprotein...post: Peptide-assisted genome editing for primary cells Monomeric StayGold variants AAV.CAP-Mac for systemic...antibodies Peptide-assisted genome editing for primary cells By Susanna Stroik Even with the advances in CRISPR...CRISPR technology over the past decade, primary cells remain difficult to genetically edit due to toxicity...toxicity from electroporation or poor cellular entry with other transfection methods. The Junwei Shi Lab addressed...Genome Editing (PAGE). They fused the Cas enzyme to cell-penetrating peptides and co-incubate it with an ...

  6. CRISPR 101: Ribonucleoprotein (RNP) Delivery

    Type
    Blog Post
    ...PMID: 27151215. PubMed Central PMCID: PMC4938170. 2. Zuris, John A., et al. "Cationic lipid-mediated delivery...can often be used in cells that are difficult to transfect, such as primary cells. Using RNPs can also...interest. This is commonly performed by transfecting cells with a plasmid, such as PX459, which encodes Cas9...must be considered when using this method:     Cells must be amenable to transfection or viral transduction...protein in complex with a targeting gRNA, to your cells of interest. Cas9 RNPs are capable of cleaving genomic...single or multi-gene knockouts in a wide variety of cell types, gene editing using homology directed repair...difficulties with protein expression that occur in cells where common eukaryotic promoters (such as CMV or...

  7. Viral Vectors 101: Chemogenetics

    Type
    Blog Post
    ...Reports 9: https://doi.org/10.1038/s41598-019-41088-2 Magnus C, Lee P, Bonaventura J, Zemla R, Gomez J, ...neuroscience where specific tissues, cell types, or even subcellular regions of a neuron need to be targeted...pathways, giving rise to the same cellular responses. The cellular response set off by a DREADD depends ...disease, neuroscientists need to be able to probe cells and circuits without disrupting normal brain function...it provides a noninvasive method for manipulating cell activity, especially neuronal firing. What is chemogenetics...drugs. Once activated, chemogenetic receptors hijack cell signaling pathways and can lead to such responses...requires successfully introducing receptors to target cells. Targeted and specific delivery becomes especially...

  8. Uncovering Ancestral Bias in CRISPR Research

    Type
    Blog Post
    ... that bind to a cell’s DNA, telling the Cas machinery where to cut.      Fig. 2: Heatmap indicating...ancestry and germline sequence variation in cancer cell lines took them down unexpected paths. Though this...researchers do large-scale CRISPR screens in cancer cell lines, looking to uncover genetic dependencies of...to see if they could identify the ancestry of the cell lines (European, East Asian, Indigenous American...or South Asian) used in the screens based on the cells’ germline genetic variations. They then wanted to...with each ancestry group. Since about 90% of the cell lines used in DepMap CRISPR screens are of either...groups for 994 of the Cancer Dependency Map cancer cell lines (left). Heatmap indicating the breakdown of...

  9. Transfection for Recombinant Antibodies

    Type
    Protocol
    ...Workflow Timeline Day 1: Seed cells Day 2: Transfect cells Day 3-6: Feed cells Day 7: Harvest antibody Equipment...Pro-Tip Do not use cells that are over 30 passages. Section 2: Transfection Check the cell density and viability... (EBNA). Safety Warnings HEK293 cells are considered biosafety level 2. Please ensure that you are in ...compatible with 50 mL conical tubes Automated cell counter 37 °C, 5% CO 2 incubator with shaking platform set to...density of 0.9 x 10 6 cells/mL in a 500 mL vented flask.Incubate in a 37 °C, 5% CO 2 incubator on a shaking ...Pro-Tip Culture should be between 1.5–2 x 10 6 cells/mL with >95% viability to proceed with transfection...250 mg Benzamidine 25 mL Aprotinin saline solution (2 mg/mL) Mix well and sterilize through a 0.2 µm PES...

  10. To Each HIS Own

    Type
    Blog Post
    ...take care when using this method.      Fig 2:  A, The polyhistidine tag is a string of histidine...This can be pretty complicated given that a single cell has tens of thousands of proteins functioning in...

  11. Antibodies 101: ELISA (Enzyme-linked Immunosorbent Assay)

    Type
    Blog Post
    ... adapted to a competitive format. Figure 2: Overview of using Competitive ELISA to quantitatively...immunoassays in DNA damage and instability detection. Cell. Mol. Life Sci. 76, 4689–4704 (2019). https://doi.org...

  12. Of Myc and Men

    Type
    Blog Post
    ...promising monoclonal antibody with high specificity. (2) Narrow down the specific epitope that your antibody...For about as long as the disciplines have existed, cell and molecular biologists have been continuously ... peer into the mysterious, microscopic world of cells. The development of antibodies as tools allowed ...monoclonals would help the field explore Myc’s role in cell proliferation and tumorigenesis.  Using two different...specific for human c-myc proto-oncogene product. Mol Cell Biol 5:3610–3616. https://doi.org/10.1128/mcb.5.12.3610... and immunoglobulin heavy chain binding protein. Cell 46:291–300. https://doi.org/10.1016/0092-8674(86...signal prevents secretion of luminal ER proteins. Cell 48:899–907. https://doi.org/10.1016/0092-8674(87...

  13. Which Fluorescent Protein Should I Use?

    Type
    Blog Post
    ...system (by measuring the fluorescence intensity); and 2) to visualize the localization of the FP (fused to... localization of that biomolecule inside living cells. FPs classified by the emission color (emission .... Therefore, these can be used as “timers” for cellular processes following their activation. The four...Biosensors: Throwing color lights on molecular and cellular processes” Curr. Protein. Pept. Sci. 9(4):338....proteins and their applications in imaging living cells and tissues”  Physiol. Rev. 90:1103. Wu et. al. ... nuclear localization, and dynamics” Curr. Opin. Cell. Biol. 23:310. ...

  14. Coomassie Purity Stain of Recombinant Antibodies

    Type
    Protocol
    ...follows: Figure 2 Using the box tool, draw a box around the entire first gel lane (as in Figure 2). Select Analyze... Example for AR0018 (lane 2 in Figure 1): Sample Peak 1 (contaminant) Peak 2 (contaminant) Peak 3 (HC)...choose Use Equation . Select the Show R 2 checkbox. Pro-Tip The R 2 of the trendline should be between 0.95...Equipment Heat block 1–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel... Add 5 µL of 4X sample buffer to each sample. Add 2 µL 10X reducing agent to each sample. Spin the sample...bottom part of the gel where dye is visible. Section 2: Staining the Gel Place the gel in a plastic tray .... Figure 1 Recombinant antibody preps should have 2 clear bands at ~50 kDa and ~25 kDa corresponding to...

  15. Fluorescent Protein Guide: Empty Backbones

    Type
    Collection
    ...- Bacterial Expression mScarlet-I3 568 592 68 4.2 2 min Monomer pmScarlet-I3_C1 - Mammalian Expression...Fluorescent Protein Resources: FRET Biosensors Subcellular localization Michael Davidson Collection Blog...University of Arizona Shaner et al. : Journal of Cell Science, Vol. 120, December 2007, pp. 4247-4260 ...

  16. xCas9: Engineering a CRISPR Variant with PAM Flexibility

    Type
    Blog Post
    ...) 12 (>100) xCas9(3.7)-ABE 69 (1.4) 43 (2) 21 (3) Not examined 16 (>100) Average percent... in mammalian cells Hu et al. compared xCas9 activity with wt SpCas9 in mammalian cells, finding xCas9...NG and GAT PAMs. xCas9 3.7 Activity in Mammalian Cells Construct NGG PAM NGA PAM NGC PAM NGT... percent editing efficiencies (edit frequency in cell population measured with NGS) are shown as percentages...

  17. Mobile-CRISPRi: Bringing CRISPRi to Diverse Bacteria

    Type
    Blog Post
    ...integrates into the trnS-leu2 locus.   Figure 2: Introducing Mobile-CRISPRi into bacteria. Top: A ... acid (DAP), a component of the cell wall. Once in the recipient cell, the CRISPRi DNA (purple) flanked... is propagated during normal DNA replication and cell division. Mobile-CRISPRi does not disrupt gene function...mobilize these plasmids for transfer to recipient cells during a tri-parental mating scheme. Mobile-CRISPRi...

  18. Colony Formation Titering Assay

    Type
    Protocol
    ...for ~2 weeks. All of the cells in the untransduced (negative) control well should be killed and no colonies...with 0.1% crystal violet and counted. Figure 2: A549 cells were transduced with the indicated serial dilutions...Day 2: Replace media with fresh media containing selection reagent Days 3–14: Change media as needed Days...Cabinet 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel pipette... 200–1000 µL single channel pipette Ice bucket CO 2 incubator Pipet controller Hazardous waste container...disturb the colonies. Count the colonies for at least 2 of the dilutions. Pro-Tip The higher dilution wells...1,000 cells into each well of a 6-well dish. Prepare a batch of cells as follows: Dilute 7,000 cells into...

  19. CRISPR 101: Targeting Non-Coding RNAs with CRISPR/Cas9

    Type
    Blog Post
    ...You’ve probably heard that only 2% of our genome is made of protein-coding genes, and you might be wondering...encode proteins. They play important roles in many cellular and regulatory processes, but much of their functionality...screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites. Nat Biotechnol...non-coding RNAs with the CRISPR/Cas9 system in human cell lines. Nucleic Acids Research. 2014;43(3):e17. doi...

  20. Bacterial Toxin-antitoxin Systems as Molecular Biology Tools

    Type
    Blog Post
    ...mazF gene and a gene for Zeocin resistance (Figure 2). This MazF-ZeoR cassette is flanked by two direct...unstable, meaning that the cells must continually produce antitoxin to avoid cell death. In the case of plasmids...addiction systems” require dividing cells to retain plasmids - if a cell does not inherit plasmid due to ...will be degraded leaving the cell to be killed by the more stable toxin. Cells only survive if they retain...10% of cells while plasmids containing the plasmid addiction system were retained in 75% of cells. They...consist of a toxin which can affect a variety of cellular processes and an antitoxin that suppresses the...designated this fragment as the ccdB region (for “coupled cell division”). The encoded toxin-antitoxin system consists...

  21. Hot Plasmids - November 2023

    Type
    Blog Post
    ... vivo and in vitro applications.   Figure 2: Modular features of DIRECTED particles where the ... between living cells by TransitID. Cell, 186(15), 3307–3324.e30. doi: 10.1016/j.cell.2023.05.044. PMID... efficient prime editors. Cell, 186(18), 3983–4002.e26. doi: 10.1016/j.cell.2023.07.039. PMID: 37657419...lentivirus and MMLV gag, to specific cell types. It can also make CAR-T cell therapy more accessible by simplifying...Optogenetic proximity labeling and TransitID Tracking cellular history with protein ticker tape Teaching an old...called DIRECTED (Delivery to Intended REcipient Cells Through Envelope Design). This modular approach ...viral surface, expanding the range of targetable cell types. DIRECTED is compatible with fusogens from...

  22. Viral Vectors 101: Preparing Pooled Libraries

    Type
    Blog Post
    ...replicate, then purify the DNA.     Figure 2: A pooled library page with protocols boxed in red...transduction of cells, resulting in cell population mutated by the viral pooled library. These cells can then...transduce your cells.  Titering After generating your lentivirus, you’ll want to infect your cells with the ...of a cell being infected by only one viral particle, but it also increases the chances that a cell doesn...adequately represented in the cell population, you’ll need to infect many more cells than the number of plasmids...appropriate packaging and envelope plasmids into HEK cells allows production of lentivirus. Purification of...small aliquot is not enough to transfect producer cells, so your first step will be to amplify the library...

  23. Hot Plasmids - August 2020

    Type
    Blog Post
    ...CRISPR in human cells) is a CRISPR-Cas13 based antiviral method to degrade RNA from SARS-CoV-2 sequences and...for labeling specific structures within cells. HeLa cells were transfected with plasmids encoding...protein evolution Fluorescent tools for mammalian cells Nanobody purification CRISPRi and CRISPRa system...selection in the cytosol (instead of on the yeast extracellular surface) which enables tracking of protease ...mCitrine gene. mCherry is constitutively expressed. Cells are then cultured and FACS sorted. Image from Mateo...labeling structures and compartments in mammalian cells By Angela Abitua Genetically encoded fluorescent...or specific localization of biomolecules within cells. However, sometimes these fluorescent proteins might...

  24. New CRISPR Tools: Cas7-11 and PASTE

    Type
    Blog Post
    ...rate of 10-55% - the PASTE system.     Fig. 2: Schematic of programmable gene insertion with PASTE...no detectable effect on cell viability – meaning there was no measurable cell toxicity – using Cas7-11...such as mouse embryonic stem cells and glioblastoma cells. PASTE Development and discovery The first ...observed in non-dividing cells, meaning that this system wasn’t dependent on the cell’s intrinsic machinery...turning potential into impact. In molecular and cellular biology, this happens through the development ...exclusively on this space — developing tools for cellular targeting. Lately, they’ve been working on two... shRNA and Cas13 systems in a range of mammalian cell lines. However, in those same comparisons, the AbuGoot...

  25. Advancing Biology with Zebrafish: Genetic Tools for Developmental Studies and More

    Type
    Blog Post
    ...externally fertilized, transparent progeny (Figure 2). These features give it a leg up (or a fin up?) over...development. Single cell sequencing-based lineage tracing Recently, gene expression in all cells of the developing...proportions of otherwise normal cell types are produced, rather than novel, abnormal cell types. Sequencing-based...understanding how genes control biology (a fact celebrated by the Nobel prize committee). But as the field...applications. For example, specific tissues such as blood cells or neurons can be fluorescently labeled, allowing...divisions, and movements of each of the thousands of cells in the developing zebrafish embryo have been tracked...developing zebrafish embryo has been mapped using single cell RNA sequencing approaches (Harland, 2018). Several...

  26. Plasmids 101: Multicistronic Vectors

    Type
    Blog Post
    ...al (Nature. 1988 Jul 28;334(6180):320-5.) PubMed. 2. A segment of the 5' nontranslated region of encephalomyocarditis...them. Why Use multicistronic vectors? Detecting cells that are expressing your gene, especially if you...reporters allow you to easily screen or select for cells that are expressing your gene of interest at high...promoter, multicistronic plasmids ensure that any cells that are positive for your marker should also be...anytime you want to express multiple genes in the same cell. Although it is possible to drive co-expression ...promotes translation of the viral genome in eukaryotic cells.1,2 Since then, a variety of IRES sequences have...discovered - many from viruses, but also some from cellular mRNAs. What they all have in common is the ability...

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

    Type
    Blog Post
    ... PMID: 25184501. PubMed Central PMCID: MC4262738. 2. Doench, John G., et al. "Optimized sgRNA design to...when trying to screen a limited number of cells like primary cells or in vivo. Recently, the labs of David... cultured cells as well as more difficult conditions such as model systems and primary cells. References...with both A375 (melanoma) and AT29 (colon cancer) cells and assessed the libraries performance via the depletion...important criterion for probing a limited number of cells. Get the Brunello CRISPRko sgRNA library as pooled...subject to an increase in dsDNA breaks that can cause cell death. Thus, CRISPR inhibition (CRISPRi) libraries...study screening for vemurafenib resistance in A375 cells. The SAM system (CRISPR/Cas9 Synergistic Activation...

  28. Split Fluorescent Proteins for Studying Protein-Protein Interactions

    Type
    Blog Post
    ...split at 175 Arabidopsis homolog of the yeast TREX-2 mRNA export complex: components and anchoring nucleoporin...measuring protein expression or localization in cells, or even single-particle tracking of the bound complex...) for use with GFP(11) Multiplexed labeling of cellular proteins with split fluorescent protein tags. ...(1-10) and GFP(11) Versatile protein tagging in cells with split fluorescent protein. Kamiyama et al. ...pairs with sfCherry2(11) Multiplexed labeling of cellular proteins with split fluorescent protein tags. ...tracking of protein-protein interactions in living cells. Shao S, Zhang H, Zeng Y, Li Y, Sun C, Sun Y. Commun...

  29. Design Tips for Prime Editing

    Type
    Blog Post
    ...inhibit editing (Anzalone et al., 2019). Figure 2: Starting lengths for optimizing your pegRNA primer...editing systems by manipulating cellular determinants of editing outcomes. Cell, 184(22), 5635-5652.e29. https...Guide RNA and Target DNA. Cell, 156(5), 935–949. https://doi.org/10.1016/j.cell.2014.02.001 Yan, J., Oyler-Castrillo...type of edit, genomic context, delivery strategy, cell type, and goal of the experiment. Now, onto the ...nicking of the unedited strand and encourage the cell to use the edited strand as a template during DNA...also allows certain unintended edits to escape cellular surveillance. To limit unintended incorporation...https://doi.org/10.1016/j.cell.2021.09.018 Nelson, J. W., Randolph, P. B., Shen, S. P., Everette, K. A...

  30. What is Polymerase Chain Reaction (PCR)

    Type
    Protocol
    ...PCR tube Ice Bucket 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 ... reagents on ice): 2 μL Template DNA (10 ng-500 ng) 5 μl 10X Taq buffer with MgCl 2 1 μl dNTP mix (10 ...normally sterile dH 2 O. To make a 100uM stock of any primer, add a number of µl of dH 2 O equal to the number...annealing temperature step-wise by 1-2°C. The rate of DNA synthesis is ~1-2 kb/min. The extension time can ...work adequately. Divalent cations such as Mg 2+ and Mn 2+ stabilize the buffer solution. These cations...(PCR). Basic PCR Program Initial Denaturation for 2 minutes at 94°C: This initiation step heats the double...target DNA strand accurately and rapidly. Repeat steps 2-4 25-30 times. Final Extension for 5 minutes at 72...

  31. Kit Free RNA Extraction

    Type
    Protocol
    ...RNAzol®, QIAzol® (for Protocol Option #2) Water-saturated Phenol 2 M Sodium Acetate pH 4 Chloroform/Isoamyl...to the Option #2 - TRIzol® Protocol section below. Homogenize or lyse tissues or cells in Solution D. ...those in -80°C. Option #2 - TRIzol® Protocol Homogenize or lyse tissues or cells in TRIzol® or a similar....5% (wt/vol) N-laurosylsarcosine (Sarkosyl) 0.1 M 2-mercaptoethanol TRIzol® or similar product such as...following sequentially to 1 mL of lysate: Add 0.1 mL of 2 M sodium acetate (pH 4.0), mix thoroughly by inversion...by hand for 10 seconds. Incubate the sample(s) for 2-3 minutes on ice and centrifuge for 15 minutes at ... D per 100 mg of cells. For cultured cells: use 1 mL of Solution D per 1 X 10 7 cells. Allow sample(s)...

  32. Degrading DNA with Cascade-Cas3

    Type
    Blog Post
    ...systems utilize with type I-F Cascades. Figure 2: Cascade complex from CRISPR type I-E. Created with...unwinding steps. Molecular Cell, 70(3), 385-394.e3. https://doi.org/10.1016/j.molcel.2018.03.031 Xiao, Y., ...CRISPR Cascade. Molecular Cell, 83(5), 746-758.e5. https://doi.org/10.1016/j.molcel.2023.01.024 Morisaka, ...there isn’t a template strand left behind that the cell can use to repair. Figure 3: General overview...broad and unidirectional genome editing in human cells. Nature Communications, 10(1). https://doi.org/10.1038...CRISPR–Cas systems for genome engineering in human cells. Nature Biotechnology, 37(12), 1471–1477. https:...

  33. Tips from the Repository Trenches: Using Barcodes to Track Samples

    Type
    Blog Post
    ...that numbers are not switched around or misread.  2. Track the container - It is just as important to ... reader and an Excel spreadsheet. Start by scanning the sample barcodes into the cells in the same order...using an electric charge to help the DNA enter the cells. Some samples require a reduced antibiotic concentration...concentration or need to be transformed into different cells than were originally requested. Most of our samples...tubes and use this ID to name the corresponding Excel sheet.3. Be specific  - When assigning a physical...storage process - from deposited DNA to transformed cells and finally to bacterial glycerol stocks that are...

  34. AAV Purification by Iodixanol Gradient Ultracentrifugation

    Type
    Protocol
    ...purification. Workflow Timeline Day 1: Purify Day 2: Buffer exchange and concentration Note: Both steps... 7.4 1X PBS-MK buffer 100X Pluronic-F68 NaCl MgCl 2 KCl Centrifugal filter units (MWCO 100 kDa) Reagent...PBS-MK buffer Dissolve 5.84 g of NaCl, 26.3 mg of MgCl 2 and 14.91 mg of KCl in 1× PBS in a final volume of...at 4 °C. 1X PBS-MK buffer Dissolve 26.3 mg of MgCl 2 , and 14.91 mg of KCl in 1× PBS in a final volume ...(C) (formulation buffer) Add 5 mL of Buffer B and 2 mL of 5 M NaCl to 43 mL PBS Procedure Preparation ...need more time, you can alternatively centrifuge for 2 h at 200,000 x g at 18 °C. Carefully take the QuickSeal...interface of the 60% and 40% gradient (see Figure 2) with an 18 ga needle. Place the first microcentrifuge...

  36. Corralling and Cornering the Goldilocks Antibody

    Type
    Blog Post
    ...non-specific binding in our IHC condition.   Fig. 2: Schematic of the goldilocks antibody selection process... Innovative Neurotechnologies (BRAIN) Initiative Cell Census Network, the Wu lab will be screening over...

  38. Save Time with Transient Plant Leaf Transformations

    Type
    Blog Post
    ...GUS-staining is a very useful visual reporter (Figure 2). The GUS enzyme cleaves a substrate, which will then...roots, as functional chloroplasts and leaf specific cell types are missing in roots. The development of transgenic...transformation” usual means that neither a stable cell line or plant is regenerated from the transformed...with the gene of interest is transferred into the cells. But for C. roseus, Agrobacterium infiltration into...fused to a protein of interest to elucidate the subcellular localization of your protein of interest. Find...Intrinsic GUS-like activities in seed plants." Plant cell reports 9.1 (1990): 1-5. PubMed PMID: 24226366. ...

  39. To Codon Optimize or Not: That is the Question

    Type
    Blog Post
    ...Hoogsten base pairs and wobble pairs.  Figure 2: (A) The codon wheel depicts the 64 codons that encodes...mammalian cells. This was the first codon optimized membrane protein expressed in mammalian cells. In this...sequence. That means there are 61 possible tRNAs. Cells however, may not express all 61 of these tRNAs and... are also optimized for expression in mammalian cells. For example, Henry Lester’s lab optimized the C...Caenorhabditis elegans GluCl ion channel genes for mammalian cells dramatically improves expression levels. Journal...

  40. Plasmids 101: Codon usage bias

    Type
    Blog Post
    ...PMID: 18478103. PubMed Central PMCID: PMC2364656. 2. Dittmar, Kimberly A., et al. "Selective charging ...reasons include: Metabolic pressures - it takes cellular resources to produce tRNAs that recognize different...mismatched translation rates and potential effects on cell growth, even hosts producing alternative tRNAs may...inclusion bodies that will generally segregate with the cell pellet during purification procedures. Even if you...a means to fine-tune gene expression." Molecular cell 59.2 (2015): 149-161. PubMed PMID: 26186290. PubMed...

  41. Course-Based Undergraduate Research Experiences (CUREs)

    Type
    Blog Post
    ...that view (Callahan et al., 2022).     Fig. 2: Students in an MDH CURE replaced select residues ...identifying new genes required for stem and progenitor cell maintenance in planarians in Developmental Biology... his teaching style, with the former offering excellent training in improvisation and the latter making...junction complex is required for stem and progenitor cell maintenance in planarians. Developmental Biology...

  42. Transferable Skills Guide: Career Planning Resources

    Type
    Blog Post
    ...American Society for Cell Biology (ASCB) or the American Chemical Society (ACS) are just 2 examples of organizations...for the position. This post on Business Insider nicely summarizes good job fair practice. Career pages..., there is an etiquette that you should follow, nicely summarized in this post on Forbes.com, and here...

  43. Protocols for Molecular Biology, Plasmid Cloning, and Viral Preps

    Type
    Protocol
    ...BSL-1 and BSL-2 Labs Learn how to best protect yourself when working in BSL-1 and BSL-2 labs Watch the...Two (BSL-1 and BSL-2) Safety measures for laboratories operating at BSL-1 and BSL-2 Watch the Video! Water...Generating Stable Cell Lines with Lentivirus Genomically integrate your DNA sequence into a cell line Isolating...Isolating a Monoclonal Cell Population by Limiting Dilution Generate monoclonal cell lines from a polyclonal...polyclonal pool of stable cells AAV Production in HEK293 Cells Produce adeno-associated virus to deliver your...Extraction Without a Kit Extract RNA from tissues or cells without a commercial kit Plasmid Cloning Name Description...Transfection Introduce plasmid DNA to mammalian cells Lentivirus Production Produce lentivirus with a ...

  44. Protocol - Bacterial Transformation

    Type
    Protocol
    ...each transformation tube by placing the bottom 1/2 to 2/3 of the tube into a 42°C water bath for 30-60 ...depending on the competent cells you are using). Put the tubes back on ice for 2 min. Add 250-1,000 μl LB...the competent cells in your hand instead of on ice Reduce step 4 from 20 - 30 mins to 2 mins on ice before...commonly referred to as 'competent cells.' Many companies sell competent cells, which come frozen and are prepared...that came with your competent cells. Pro-Tips Commercial competent cells range significantly in their ...SOC media Competent cells DNA you'd like to transform Procedure Take competent cells out of -80°C and thaw...use electro-competent cells. Instead of relying on the heat-shock to cause the cells to take up the DNA,...

  45. Antibody Validation Using the Indirect ELISA Method

    Type
    Protocol
    ...vapors. Workflow Timeline Day 1: Antigen Coating Day 2: Blocking Day 3: Primary antibody incubation Day 4...with 96-well plates 1–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...serial dilutions of the purified antigen as follows: 2 ng/µL : Add 100 µL of 20 ng/µL stock into 900 µL PBS...microfuge tube and vortex. 1 ng/µL : Add 450 µL of 2 ng/µL stock into 450 uL PBS in a microfuge tube and...37 °C for 30 min , or overnight at 4 °C . Section 2: Block the plate Prepare the wash buffer (0.05% Tween...incubate on a microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section...

  46. Bioinformatics at Addgene

    Type
    Blog Post
    ...the necessary steps to perform:      Fig. 2: Quick-stepping to a FASTA file   With our outline...mission and our own, as both address the need to accelerate science using different approaches. We were also...evolved into a single, organized pipeline and accelerated the processing of our sequencing data. However...

  47. Immunocytochemistry

    Type
    Protocol
    ...HeLa cells per well. Allow the HeLa cells to grow to the desired density before labeling. Section 2: Fixing...coated coverslips HeLa cells 24-well plate 4% Paraformaldehyde 5 mg/mL 4′,6-diamidino-2-phenylindole (DAPI...Fluorescent microscope 0.5–10 µL single channel pipette 2–20 µL single channel pipette 20–200 µL single channel...of the diluted antibody to the wells and incubate 2 h at RT . Remove the primary antibody and dispose ...detect antigens in cells. Here we describe the basic steps for fixing and labeling cells in culture with ...2022 Workflow Timeline Day 1: Seed cells Day 3-4: Fix and label cells Equipment Pipette controller Pipette... Seeding cells Place a sterile poly-D-lysine coated coverslip in each well of a 24-well cell culture treated...

  49. Protocol - How to Inoculate a Bacterial Culture

    Type
    Protocol
    ...mL glass bottle: 4 g NaCl 4 g Tryptone 2 g Yeast Extract and dH 2 O to 400 mL Note: If your lab has pre-mixed... to do a mini-prep you will usually want to start 2 mL in a falcon tube, but for larger preps you might...might want to use as much as a liter of LB in a 2 L Erlenmeyer flask. Using a sterile pipette tip or toothpick...bacterial cell. Large plasmids usually have a low copy number (approximately one or two copies per cell) and... can be present in large numbers, 50 or more per cell and have a high copy number. High copy number plasmids...increase this to 350 - 400 rpm to obtain a higher cell density. Reference Page Top Index...
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