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Showing: 31 - 60 of 749 results

  1. Replacing Paper: Tips for Choosing an Electronic lab Notebook

    Type
    Blog Post
    Published
    July 10, 2018, 1:08 p.m.
    ...and developed the SciNote Electronic Lab Notebook. Implementing an electronic lab notebook starts with ...should have functionalities such as: electronic signatures, electronic witnessing and audit trails. How ...write things down when doing scientific research? Electronic lab notebooks and researcher's needs The familiar...traceability and reproducibility of research. Electronic lab notebooks (ELNs) are here to make lab work...This includes financial outlay, staff hours, troubleshooting, and the fact that long-term use is likely ...traceable. However, switching from paper to an electronic lab notebook requires time and knowledge. In ...recent article, we translated our knowledge of electronic lab notebooks into 9 questions that will help...

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

    Type
    Blog Post
    Published
    April 8, 2025, 1:15 p.m.
    ...loading controls are equally strong, so the normalized value simply reports which POI band is stronger. The...loading control is not perturbed by the experiment The loading control is supposed to control for, well...western blots benefit from a positive control — a loading control — that you know is present in every lane...transfer. Figure 1: A loading control is a type of positive control necessary to demonstrate a POI’s...loading control: the intensity of each POI band is divided by the intensity of the loading control in the...with loading controls. Under each lane, the fraction of the POI band over the loading control is shown. ...loading control. First, let’s explore why these assumptions are key to using loading controls accurately...

  3. Viral Vectors 101: AAV Serotypes and Tissue Tropism

    Type
    Blog Post
    Published
    Jan. 16, 2025, 2:15 p.m.
    ...choice for targeting astroglia. It has additionally been reported to have tropism for liver, vascular ... It can transduce neurons for retrograde labeling, as well as astrocytes in the CNS (Han et al., 2023)...following table summarizes tissue tropism as we currently understand it. Tropism depends on many variables, ...Table 3: AAV tissue tropism. To target... Try using... CNS AAV1, AAV2, AAV2-retro (AAVrg), AAV5, AAV8...enables efficient retrograde targeting of projection neurons and enhances astrocyte-directed transduction...the opposite: AAV serotypes with varying tissue tropism, allowing them to target particular cell types....commonly used serotypes in this post. However, tissue tropism varies both between species and even between strains...

  4. Viral Vectors 101: Gamma-Retroviral Packaging Systems

    Type
    Blog Post
    Published
    Aug. 6, 2024, 1:15 p.m.
    ...The widespread use of γ-retroviruses (gamma-retroviruses) in cancer and stem cell research has prompted...flexibility to pseudotype γ-retrovirus using different envelopes to modify tropism. Briefly, different envelope...either an ecotropic envelope, Phoenix-ECO, (for infection of mouse and rat cells), or an amphotropic envelope...prototypical gamma-retrovirus.    Frequently asked questions about γ-retroviral vectors How do engineered...MoMLV and MSCV for γ-retrovirus; HIV for lentivirus). Additionally, γ-retroviruses can only transduce dividing... Are γ-retroviruses self-inactivating (SIN)? They can be. Plasmids that encode SIN γ-retrovirus lack most...blog Viral Vector Elements The Retroviral Life Cycle Popular Retroviral Vectors and Their Use in Research...

  5. Plasmids 101: NGS Quality Control for Pooled Libraries

    Type
    Blog Post
    Published
    Oct. 26, 2017, 1:59 p.m.
    ...Addgene uses electrocompetent Stbl4 cells when performing amplifications, and we use electroporation to ensure...library, Addgene usually does four to eight electroporation reactions using a total of ~400ng of library...

  6. AAV Vector Quality Control: Going the Extra Mile with NGS

    Type
    Blog Post
    Published
    Sept. 12, 2017, 1:44 p.m.
    ...highest quality, we undertake a rigorous quality control process. In 2016, we launched our viral service...our scientists perform all the standard quality control assays for AAVs. These steps include measuring ...Addgene is also conducting more rigorous quality control, including next generation sequencing (NGS) of ...VGS). Why should you care about viral quality control? AAV vectors are produced by scientists - not robots...Lecomte et al., 2015). This higher level of quality control is recommended but not yet required for clinical-grade...addgene.org to answer your questions about our quality control process! The images in this blog post were created...2011) Vector Characterization Methods for Quality Control Testing of Recombinant Adeno-Associated Viruses...

  7. What’s in Your Tube?: A Quick Guide to Measuring DNA by Spectrophotometry

    Type
    Blog Post
    Published
    July 1, 2025, 1:15 p.m.
    ...the go-to method is to use spectrophotometry. Microvolume spectrophotometers make it quick and easy to ... jumping in — in the world of microvolume spectrophotometers, the Thermo Scientific NanoDrop is one of...details might vary.  How it works — briefly Spectrophotometers measure how light travels through a sample...contaminants, some of which are easier to detect by spectrophotometry than others. The ones most likely to impact...making it more difficult to detect. A particularly strong absorber at 230 nm is guanidine thiocyanate (used...Phenol is a unique case, as pure phenol absorbs strongly at ~270 nm and exhibits A260/280 and A260/230 ...distinguishing between the two can be difficult by spectrophotometry. RNA tends to yield a higher A260/280 and ...

  8. Interview: Nicola Patron on Plant Synthetic Biology, MoClo, and More

    Type
    Blog Post
    Published
    June 12, 2014, 3:30 p.m.
    ...Nicola Patron is Head of Synthetic Biology at the Sainsbury Laboratory, where she often feels more like...that go on between plants and their pathogens. As Patron explains it, her work has always been focused on...Modular Cloning (MoClo) Kit. What does it include? Patron: Sainsbury has a mandate to share and educate and...Addgene: Will the MoClo tools work in any plant? Patron: The MoClo kit is entirely for dicots, not grasses...tips for those looking to get started with MoClo? Patron: The parts will be familiar to most plant scientists...there other tools you are working on for plants? Patron: Our lab and others at the Sainsbury are working... of engaging about science in that way for you? Patron: I started my Twitter account recently inspired...

  10. Plasmids 101: In-house NGS Plasmid Quality Control Now at Addgene

    Type
    Blog Post
    Published
    April 6, 2021, 12:30 p.m.
    ...and last updated Apr 6, 2021 by Will Arnold. Introducing NGS in 2017 for all incoming plasmids was a big...Addgene - we had used Sanger sequencing for quality control for over ten years. Now, thanks to a generous donation...quantification of various intermediate steps, a sizing electrophoretic gel, and qPCR quantification of the final libraries...The first step we take when performing quality control of newly deposited plasmids is to align the NGS... by the depositing lab, we call these “quality control (QC) issues.” We then ask the depositing laboratory...

  11. Plasmids 101: Walkthrough of Addgene’s Snapgene-Powered Quality Control Process

    Type
    Blog Post
    Published
    Feb. 1, 2018, 3:07 p.m.
    ...talked a lot about the quality control process at Addgene by introducing our new sequencing partner seqWell...generation sequencing results to perform quality control on deposited plasmids. We’ve also talked about ...interface. We regularly use Snapgene for our quality control process because of its expansive feature library... main ways we use SnapGene for plasmid quality control are: To identify important plasmid features ...Identifying plasmid features While our quality control process for incoming plasmids can vary based on... quality control... plasmid quality control...

  12. Export Control Regulations Lifted for Plasmids Containing Rabies and VSV-G Genes

    Type
    Blog Post
    Published
    Feb. 6, 2025, 2:15 p.m.
    ...pUMVC (MuLV retroviral). Bob Weinberg pMD2.G VSV-G envelope expressing plasmid Didier Trono pVSV-G...plasmids. This is because in 2024 certain export control exceptions were made by the US Bureau of Industry...Industry and Security, pursuant to the international controls prescribed by the Australia Group, for these plasmids...Envelope protein for producing lentiviral and MuLV retroviral particles. Use in conjunction with a packaging...researchers in countries with applicable export control exceptions for rabies and VSV-G genes, plasmids...strive to keep our information updated, export controls and regulations are complex and may change suddenly...

  13. Teaching an Old DOG New Tricks: Controlling Protein Activity with GFP

    Type
    Blog Post
    Published
    Nov. 24, 2015, 3:30 p.m.
    ... just label cells. If GFP could be co-opted to control gene expression, he could then selectively manipulate...promoters. Moving to an in vivo system, Tang et al. electroporated T-DDOGs, GFP, and a reporter construct into...TdTomato, whose expression was absent without electroporated GFP. They then tested T-DDOGs with two mouse...robust and specific. When tested in retinal electroporation studies, DsRed reporter expression was induced...

  14. A New Optogenetic Tool Based on AraC Controls Gene Expression with Blue Light

    Type
    Blog Post
    Published
    June 17, 2021, 1:17 p.m.
    ...Their system is based on the AraC protein that controls transcription from the PBAD promoter. “AraC is... promoter. While none of the constructs gave as strong a signal as with the wild-type AraC, it still reached... of interest, under the pBAD promoter, can be controlled with light without the need of any cloning. ...

  15. Controlling for Off-target Effects with a New Genome-wide CRISPR Screen Design

    Type
    Blog Post
    Published
    Sept. 13, 2018, 1:55 p.m.
    ...an appropriate control? Many CRISPR screens use non-targeting guides as negative controls. Non-targeting...are well characterized and strategies exist to control for these faults. However, it’s still unclear if...screens and how best to design these screens to controls for flaws. Recently the Bassik Lab at Stanford...site in the genome. This means that while they control for many of the effects of gRNA and Cas9 expression...detecting Cas9 cutting. Safe targeting guides better control for non-specific toxicity than non-targeting guides... a coding region. Targeting these sites should control for the effects of guide expression and dsDNA breaks... the genome, and are commonly used as negative controls for genome-wide CRISPR screens; and 3) safe-targeting...

  18. Quick Guide to Working with Drosophila Part 2: Controlling Gene Expression in Flies with Gal4/UAS

    Type
    Blog Post
    Published
    July 21, 2017, 12:48 p.m.
    ...systematically study gene function with temporal control and cell-type specificity! The Gal4/UAS system...you mate your fly line containing YFG under the control of the UAS with a fly line expressing Gal4 in your...promoter each time a new hypothesis is tested. Controlling multiple genes with Gal4/UAS system One can also...to drive expression of multiple genes under the control of UAS enhancer sequences. For example, if you ...them individually or simultaneously. Do note for control purposes that there are dosage effects. For example...

  19. Pooled CRISPR Libraries Offer Genome-Wide Control for Large-Scale Functional Screens

    Type
    Blog Post
    Published
    Feb. 24, 2015, 7:50 p.m.
    ...for gene control over a 1,000-fold range. Weissman explained that there are two levels of control in the...technology has changed how scientists edit and control genes, but according to the Broad Institute's Silvana...screening In October, Jonathan Weissman’s lab introduced additional pooled CRISPR libraries, also available...abilities to turn genes on and off,” he said. “Some are strong and some more moderate; there is an allelic series...size of the backbone, transformations require electroporation. References & Further Reading: For more ...Epub 2014 Dec 10. Genome-Scale CRISPR-Mediated Control of Gene Repression and Activation. Gilbert LA, ...

  20. Viral Vectors 101: Systemic Capsids

    Type
    Blog Post
    Published
    Oct. 12, 2023, 1:15 p.m.
    ...specificity, or retrograde transport in neurons. (b) Genetic cargo can be customized to control transgene expression... Capsid Published Tropism Model Organism Mouse Strain-Specific Tropism?  Reference AAV-...and astrocytes Mice No (LY6C1)   Hanlon et al., 2019 AAV-BI-28 CNS: neurons, astrocytes, oligodendrocytes...-Specific Tropism?" column. For a complete list of mice strains with LY6A-dependent tropism, see S3 table...derived from AAV9 using iTransduce, shows tropism toward astrocytes and neurons, with greatly increased efficacy... appears to show a broader tropism with a potential bias toward astrocytes (Chuapoco et al., 2023). Additionally...systemic AAVs with endothelial-specific tropism in rodents and broad tropism in primates. Nature Communications...

  21. Using AAV for Neuronal Tracing

    Type
    Blog Post
    Published
    Aug. 9, 2018, 1:04 p.m.
    ...developed a new AAV variant, rAAV2-retro, that migrates retrogradely at higher rates than any other AAV... AAV2-retro vectors with a variety of transgene/promoter combinations. Check out our retrograde AAV blog...requires both antero- and retrograde labelling, synaptic restriction as well as controlled monosynaptic and polysynaptic...Selective retrograde transsynaptic transfer of a protein, tetanus toxin, subsequent to its retrograde axonal...bodies to the terminals of their axons; while retrograde tracing outlines neurons in the opposite direction...axons to their cell bodies. Anterograde and retrograde tracing take advantage of existing transport ...myosin, and enzymes for transmitter synthesis. Retrograde transport is used for transport of endocytosed...

  22. Plasmids 101: The protein expression toolbox

    Type
    Blog Post
    Published
    Feb. 16, 2023, 7:05 p.m.
    ... What kind of control? On, off, tunable, and/or inducible? How long do you need control? Will temporary...temporary control (a few days) be enough, or do you want a permanent control mechanism? Where is the control...transcription instead of introducing a DNA double strand break. Transient introduction of CRISPRi or inducible...vector! On/off control all the time If your experimental system requires tunable control - or maybe you...price of tunable control These systems can be easily implemented for exogenously introduced proteins, as ...prior to introduction of the construct into cells. However, if you would like tunable control of an endogenously...forever? You don’t need to resort to the force to control protein expression in mammalian cells: easy to ...

  23. Viral Vectors 101: Types of viruses

    Type
    Blog Post
    Published
    June 6, 2023, 1:15 p.m.
    ...viruses: gamma-retrovirus, lentivirus, adenovirus, and adeno-associated virus. Gamma-retrovirus Retroviruses...type of gamma-retrovirus is Murine Leukemia virus (MLV). Naturally isolated gamma-retroviruses have been ...Lentivirus is a type of retrovirus and shares many common features with gamma-retrovirus including genome architecture... nucleus as gamma-retroviruses do. Fun fact: Since lentivirus and gamma-retrovirus are so closely related...expression (Robinson, 1982). To add insult to injury, retroviruses generally have moderate to high immunogenicity...immune response in the host. So why use a gamma-retrovirus in the lab? Well, they are essentially evolutionarily... types with a high efficiency. However, gamma-retrovirus infection is restricted to dividing cells only...

  24. Advanced Uses of Cre-lox and Flp-FRT - A Neuroscientist’s View

    Type
    Blog Post
    Published
    Oct. 19, 2017, 1:54 p.m.
    ...A common means to achieve ligand control is to fuse a mutant estrogen receptor (ER) ligand binding domain...PSD95-mcherry) under the control of Cre recombinase. After in-utero electroporation, this plasmid mix results...tracking for instance) can be controlled with FLP-FRT. For further temporal control, Tamoxifen inducible Cre...system, but it only gives you a single level of control without modification - either Cre is there or it...for site specific (and often cell type specific) control of DNA recombination and gene expression can be...addition, a variety of means to spatiotemporally control FLP and Cre expression have been developed. Read...FLP and Cre enable additional levels of genetic control. What is FLP FRT and how does it work? FLP recombinase...

  25. Antibodies 101: Flow Cytometry

    Type
    Blog Post
    Published
    July 20, 2021, 1:15 p.m.
    ...Include proper controls To ensure accurate data analysis, include a complete set of controls. Ideally, every...will have a negative control that does not express the target and a positive control that does. The negative...of experiments easier and faster to perform. Introduction to flow cytometry Flow cytometry allows users... (3): An optics system A fluidics system An electronics system  Cells are first resuspended in a pressurized...fluorophore or fluorochrome-stained protein will emit a stronger photon than a cell expressing low levels of the... further the shift of a cell from the negative control, the higher the level of expression. A routine ...triton-x, saponin, or tween. Take care when using strong detergents like triton-x and tween as extended ...

  26. Important Considerations When Using AAVs

    Type
    Blog Post
    Published
    June 13, 2017, 2:30 p.m.
    ...larger amount of vector using a serotype with strong CNS tropism (4). If axonal transport is not desirable...receptor physiology and the effects of metabotropic and ionotropic influences on overall cell function. ... thanks to our guest blogger Katrina Armstrong. Katrina Armstrong is a Neurophysiology Msc Student at ...post was contributed by guest blogger Katrina Armstrong, a Neurophysiology Msc Student at the University...known is that different serotypes have different tropisms but, as you can see, AAVs of each serotype can... asking questions!  Table 1: AAV Serotypes and Tropism Experiment  Detection Method CNS AAV1, AAV2...>AAV8>AAV6>AAV5>AAV1>AAV2 This preferential tropism for different brain regions is largely due the ...

  27. Delivery Methods for Generating iPSCs

    Type
    Blog Post
    Published
    April 17, 2018, 1:37 p.m.
    ...Moloney murine leukemia virus (MMLV)-derived retroviruses. Retroviral vectors, such as pMXs or pMSCV, infect...delivery methods. Retrovirus has a cloning capacity of ~8 kb, so either a single polycistronic vector or multiple...10 kb) than other retroviral vectors. This makes it possible to use a polycistronic cassette that expresses...Mechanism Efficiency Safety Pros Cons MMLV-derived Retrovirus Integrating High Low Efficient and Stable Genome...Need to sequence cells to verify excision didn’t introduce mutations Adenovirus Non-integrating Low Medium...required for translational use. Viral Vectors Retroviral: Some of the first iPSCs were generated with ... corresponding endogenous pluripotency genes. Retroviral vectors are prone to incomplete silencing of ...

  28. Plasmids 101: Stringent Regulation of Replication

    Type
    Blog Post
    Published
    June 20, 2024, 1:15 p.m.
    ...DNA.  Plasmids are said to be under stringent control when they depend on the host cell’s initiation ...replication independently are said to be under relaxed control, as they only require the host's replication machinery...replication mechanism of plasmids under stringent control, such as pSC101. DNA replication and regulation...round of replication. Initiation is the primary control point of DNA replication in bacteria, so the process... the bacterial chromosome and for stringently controlled plasmids. Binding of IHF (green) redistributes...binding is necessary to initiate replication, controlling the amount of active DnaA available is one of...source, ATP binding is one more way for the cell to control DNA replication. A variety of other proteins also...

  29. Plasmids 101: The Promoter Region – Let's Go!

    Type
    Blog Post
    Published
    April 3, 2014, 8:05 p.m.
    ...target genes. The sequence of the promoter region controls the binding of the RNA polymerase and transcription...promoters is commonly employed in lentiviral and retroviral constructs and we will discuss these in a future... all the time while others are more carefully controlled. Regulated promoters might act only in certain... light. In the cell, promoters themselves are controlled by still other regulatory factors: enhancers,...Additional considerations CMV General expression mRNA Strong mammalian expression promoter from the human cytomegalovirus...some cell types. EF1a General expression mRNA Strong mammalian expression from human elongation factor...promoter hybrids. CAG General expression mRNA Strong hybrid mammalian promoter Constitutive  Contains...

  30. 27 Hot Plasmids from 2016

    Type
    Blog Post
    Published
    Dec. 22, 2016, 3:03 p.m.
    ... under the control of the cI857 temperature-sensitive repressor. The temperature-controlled expression...activity in the Co-IP compared of a control Co-IP indicates stronger interaction. The system has been demonstrated...DECEMBER 2016 Restoring yeast prototrophy with plasmids Though auxotrophic markers are used frequently ...complement strains with multiple auxotrophies or using different auxotrophic backgrounds can lead to disparate...their Yeast Prototrophy Kit, a set of plasmids designed to complement unused auxotrophies in Saccharomyces... at a dose and duration sufficient to kill all control cells and surviving “mutant” colonies were deep...GTPases involved in cell signaling pathways that control many cellular processes such as differentiation...
Showing: 31 - 60 of 749 results