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We narrowed to 605 results for: RAN-1

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  1. Tips for CRISPR Gene Editing in Mice

    Type
    Blog Post
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    June 28, 2016, 10:59 a.m.
    ...screening for the best gRNA Figure 1: Validation of double strand break (DSB) mediated homology dependent...as control (Cetn1/sgRNA1), 2; darker than control, 1; very dark). Brighter fluorescence indicates a more...and greatest in scientific content.      References 1. Cho, Seung Woo, et al. "Analysis of off-target effects...repair (HR, homologous recombination; SSA, single strand annealing) took place and reconstituted the EGFP...site. (c) The pCAG-EGxxFP target plasmid was co-transfected with pX330 plasmids containing sgRNA sequences...efficacy of your gRNAs is then a simple matter of transfecting HEK293T cells with your modified pCAG-EGXXFP...fluorescence, the better that gRNA is at causing a double stranded break at the target site, allowing the two halves...
  2. Important Considerations in Optogenetics Behavioral Experiments

    Type
    Blog Post
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    Oct. 1, 2015, 2:30 p.m.
    ...derekpsimonphd.    References 1. Stuber, Garret D., et al. "Excitatory transmission from the amygdala to nucleus...animal will repeatedly nose-poke at the active hole [1, 2].    Place preference/place aversion also models... can be explained primarily in two different ways 1) your hypothesis is wrong or 2) there is some problem...examples I’m personally interested in include: intracranial self-stimulation (ICSS) and place preference...(viral expression, efficiency of laser light transmission to the target neurons, appropriate stimulation...optogenetics is performed in this species. However, transgenic rat technology is improving and may one day reach... reach the sophistication of mouse transgenics. Everything that has been described is intended for the...
  3. An Addgene Summer Internship

    Type
    Blog Post
    Published
    Aug. 8, 2024, 7:22 p.m.
    ...two weeks, we were able to obtain eight clones. We ran digested and undigested samples of the clones on ...intern to complete and present on.    Figure 1: Addgene's summer interns presenting their capstone...and I wouldn’t want it any other way. Toj Toj-Villagran is a rising senior in high school who just completed...
  4. Hassle-free 96-well Format Epitope Tagging Using Cas9 Ribonucleoprotein

    Type
    Blog Post
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    June 28, 2018, 3:01 p.m.
    ...References 1. Bressan RB, Dewari PS, Kalantzaki M, Gangoso E, Matjusaitis M, Garcia-Diaz C, Blin C, Grant V, ...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....binding to the template strand (strand used by RNA polymerase II for transcription) are more effective for...complex. This complete transfection mix, i.e. csRNP plus ssODN, is then transfected into stem cells. Cells... were transfected in 96-well format using an Amaxa Shuttle device; 5 days after the transfection, the ...
  5. Quantifying DNA? Here are Five DNA Quantification Methods to Consider

    Type
    Blog Post
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    July 21, 2020, 1:15 p.m.
    ...automated. Unlike gel electrophoresis, you only need 1-2 ul of sample and the run time is just a few minutes...quantified first before larger fragments. Figure 1: The quantification of a RNA ladder using capillary...This method involves measuring the absorbance/transmission of light through a liquid to determine the concentration...spectrophotometer measures these absorbances using UV-transparent cuvettes. First you measure the absorbance of...PicoGreen or SYBRGreen, are specific for double strand DNA (as compared to spectrophotometric methods ...However, the measurements are made in the visible range and can be read with a standard ELISA reader if ...
  6. QC Sequencing Technologies at Addgene

    Type
    Blog Post
    Published
    July 15, 2025, 1:15 p.m.
    ...is recognized and recorded as a DNA strand is synthesized (Figure 1). Sanger sequencing requires four separate...trace” file to determine the sequence. Figure 1: Overview of Sanger sequencing technology. Created...Technologies. Current Protocols in Molecular Biology, 122(1). https://doi.org/10.1002/cpmb.59  Wang, Y., Zhao,...“nanopores” that are attached to the flow cell membrane and have an electrical current running through...
  7. New Norepinephrine Indicators: nLightG and nLightR

    Type
    Blog Post
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    Dec. 12, 2023, 2:15 p.m.
    ...nLightR (Kagiampaki et al., 2023).     Figure 1: Properties of nLightG and nLightR. A, Structural ...Article 6. https://doi.org/10.1038/s41592-022-01488-1 More resources on the Addgene blog Fluorescent proteins...generating variants with improved affinity and dynamic range of both nLightG and nLightR, and may soon have a...
  8. How to Negotiate a Successful Lab Start Up Budget

    Type
    Blog Post
    Published
    Sept. 1, 2015, 2:30 p.m.
    ...your budget, you should take the following steps: 1. Define your timeline Start with your thesis in mind...these decisions in the following order of importance: 1. First make decisions based on experimental timelines... several factors aren’t taken into consideration. 1. Always focus on the science When negotiating finances...will take to secure your first (R-level) research grant.  Set your timeline from there, then start identifying...techs’ financial books.  To find personnel salary ranges, go to online job information sites (like, indeed.com...
  9. Antibodies 101: Avoiding the Mouse on Mouse Mess in IHC

    Type
    Blog Post
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    Sept. 21, 2023, 1:15 p.m.
    ...following three strategies might help!     Figure 1: Species on species staining can be a problem because...same host species as your secondary antibody since 1) your secondary should not recognize Fab fragments...References and Resources References Goodpaster T, Randolph-Habecker J (2013). A Flexible Mouse-On-Mouse Immunohistochemical...
  10. Scientific Peer-review: Providing Critical and Kind Feedback and Advocating for Open Science

    Type
    Blog Post
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    Aug. 8, 2019, 1:19 p.m.
    ...the journal-specific guidelines.  Peer-review step 1: The first read At the first read of the paper, I ...group of Prof. Mark Tester, where she focuses on (1) salt-induced changes in root-to-shoot ratio in Arabidopsis... discipline and reduces the dissemination of unwarranted claims or unacceptable interpretations. A good...studying plant development and abiotic stress tolerance, I have been peer-reviewing journal submissions...
  11. Plasmid Cloning by PCR

    Type
    Blog Post
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    March 29, 2016, 2:30 p.m.
    ... by PCR has error rates that range from roughly 1 per 500bp to roughly 1 per 10 million bp depending on...chance for mis-priming, so you can use a pretty wide range of annealing temperatures, but you may need to increase...
  12. Even more elegant: Single injection CRISPR/Cas9 in C. elegans

    Type
    Blog Post
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    July 7, 2015, 3:36 p.m.
    ...sequences are inserted into the genome. Figure 1: Self-excising cassette schematic from Dickinson et...):1028-34. doi: 10.1038/nmeth.2641. Epub 2013 Sep 1. PubMed. Find plasmids from this publication at ...can generate a fluorescent protein fusion, transcriptional reporter, and loss-of-function allele in just...markers. Bob Goldstein’s lab previously used a transgene selection strategy for their C. elegans/CRISPR...
  13. Revamp Your Lab Meetings With Creative Virtual Collaboration

    Type
    Blog Post
    Published
    Feb. 9, 2021, 2:15 p.m.
    ...those ideas and build from those.   Figure 1: Virtual lab meeting? No problem! You can still creatively... on it – so it is important to take breaks. After 1 hour (or another designated time) of meeting, someone... creativity are pivotal in writing publications/grants too. As described by several works (Wuchty et al...and do a quick break to chat. This can be done randomly or manually assigning which participants to group...
  14. Plasmids 101: Inducible Promoters

    Type
    Blog Post
    Published
    Jan. 18, 2018, 2:34 p.m.
    ...Hsp70, the heat shock releases heat shock factor 1 (HSF-1), which subsequently binds to heat shock elements... polymerase and transcription factors. Since the promoter region drives transcription of a target gene...promoter to induce transcription of the phage repressor cI. Repressor cI inhibits transcription from phage promoter...lag time due to the time needed for transcription/translation. Many thanks to Nicole Zurcher who contributed... the promoter, turning it ON and initiating transcription.     Negative inducible In the OFF state, the...a bound repressor protein actively prevents transcription. Once an inducer binds the repressor protein...the DNA. With the repressor protein absent, transcription is turned ON. Types of inducible promoters Chemical...
  15. Fluorescent Proteins 101: GFP Fusion Proteins - Making the Right Connection

    Type
    Blog Post
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    April 9, 2019, 1:13 p.m.
    ...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...et al., 2018). Unbiased, random insertions can be generated with a transposon-based strategy (Sheridan...fluorescent fusion proteins: random insertion of GFP with an in vitro transposition reaction." BMC neuroscience... This type of interference is known as steric hindrance. For instance, GFP may occlude catalytic sites...sites or motifs that are necessary for post-translational modifications. Despite these potential issues...
  16. Evolution of Lab Techniques

    Type
    Blog Post
    Published
    June 21, 2016, 2:30 p.m.
    ...blotting, named for its inventor Edward M. Southern (1), can be used to detect DNA identity, size, and abundance...hours at low cost with restriction mapping (Figure 1). Though some labs may prefer to send DNA directly... serotonergic system and behavior.     References 1. Southern, Edwin Mellor. "Detection of specific sequences...into smaller pieces, run on a gel, and transferred to a membrane for analysis through hybridization of ...researchers generating transgenic animals may choose Southern blotting to screen founder transgenic animals for ...techniques used to generate transgenic mouse lines, such as BAC transgenic or traditional knockins/knockouts... control sample. Southern blotting applications range from confirming the results of cloning or knockout...
  17. Viral Vectors 101: The AAV Retrograde Serotype

    Type
    Blog Post
    Published
    April 27, 2023, 1:15 p.m.
    ... There are two methods of neuronal tracing (Fig. 1): one to visualize neurons that send information to...significantly updated in 2023 by Jason Nasse.  References 1. Tervo, D. Gowanlock R., et al. "A designer AAV variant...neurons! Neurons are really cool because they can transmit information, for example, from one of the 180 ...associated virus (AAV) shows relatively low retrograde transport efficiency in most serotypes. Some viruses (e....e.g., rabies) naturally exhibit retrograde transport and can therefore perform retrograde tracing, but...efficiently infects neurons, directs high-level transgene expression, and has retrograde functionality to...genetically controlled function. By using AAVrg in Cre transgenic mouse lines, you can access and manipulate an...
  18. CRISPR Guide Update

    Type
    Blog Post
    Published
    Oct. 10, 2024, 1:15 p.m.
    ...refreshed figures made using BioRender.com. Figure 1: PRIME editing Please check out the updated guide... to use CRISPR plasmids in the lab and the vast range of possibilities at their disposal. With this update...CRISPR’s ability to make large genome edits using transposases or the Cascade-Cas3 system. We also describe...
  19. Building and Validating Recombinant Antibodies: A Case Study

    Type
    Blog Post
    Published
    April 5, 2022, 1:20 p.m.
    ...restriction digest and colony PCR.   Fig. 1: (A) Schematic of cloning, expression and validation... doi: 10.1371/journal.pone.0038313. Epub 2012 Jun 1. PMID: 22675541; PMCID: PMC3365890....-mAbs. It was recognized that an aberrant IgG light chain transcript was expressed by some of the hybridomas...heavy (blue) chains as driven by two CMV promoters (orange). Figure from Andrews, et. al., Elife  A limitation...IF-ICC.) This method was chosen because cells transfected with a plasmid to express the target antigen ...the restriction enzyme BciVI was present in the aberrant light chain sequence, but was predicted to occur...express multiple subclasses of IgG antibody. Broad range secondary antibodies are commonly used in research...
  20. Five Popular Model Organisms, Part 2

    Type
    Blog Post
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    Sept. 29, 2020, 1:15 p.m.
    ...research like metabolism. Transport, biofilms, and much more. Because of it’s BSL-1 status, E. coli can be...oxygen. Additionally, its status as a Biosafety Level 1 microbe makes it a perfect candidate for introductory...with superior performance. Nature Communications, 9(1). https://doi.org/10.1038/s41467-018-04899-x Yokoyama...plasmids for use in CRISPR and Tol2 transposase induced transgenesis.  Xenopus Xenopus, or frogs, are one...have created molecular biology tools to generate transgenic axolotls for an array of different studies. Elly...popular tools for studying frogs include egg transplants and microinjections. Injections of mRNAs, for...Arabidopsis. There are also important tips for plant transformations and the use of Arabidopsis in the classroom...
  21. Crowdfight, a Platform to Boost Scientific Collaboration During COVID-19 and Beyond

    Type
    Blog Post
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    June 1, 2021, 1:15 p.m.
    ...prompted us to create Crowdfight.   Figure 1: Crowdfight is a community of scientists who believe...collaborator for a specific task, Crowdfight will: 1) Find an expert with the right skill; 2) Confirm the...with viruses or transmissible diseases, so we were left with no choice but to quarantine in our houses,...simpler and the volunteers helped with things like translations, bibliographic searches, etc.     Expanding...we call “asymmetric collaborations.” These are transient partnerships where a researcher makes a small ...blog post about remote collaborations Read our Transferable Skills Guide article about external collaborations...
  22. Advice on Career Paths and the Green Card Process for International Researchers and Entrepreneurs

    Type
    Blog Post
    Published
    Dec. 20, 2017, 2:33 p.m.
    ...of an industry H-1B need to be aware of the April 1 deadline for companies to submit an application on...and non-profit organizations do not have the April 1 H-1B queue process or quota like industry, these organizations... National Interest Waiver. The three prongs are: (1) The foreign national’s proposed work endeavor must...-1B, you need to prove 3 threshold requirements: (1) that you have international recognition for your ...the non-profit sector are transferrable to industry such as technology transfer, communications, medical... minimum (8 U.S. Code § 1184 - Admission of nonimmigrants). The foreign worker must possess at least a...and therefore the academic H-1B visa cannot be transferred to an industry position. If you cannot get an...
  23. Generating Mouse Models Using CRISPR/Cas9

    Type
    Blog Post
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    July 12, 2016, 2:30 p.m.
    ...always guaranteed. To learn more about this process check out our Mouse Modeling blog posts: Part 1: Genetically...that better human health.     References Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu...Last updated on Oct 1, 2020 by Aliyah Weinstein. This post was contributed by guest bloggers, Wenning...3.4% of founders establishing germline transmission of the transgene, this technique is still in need of ...and chimeric adults mated to produce completely transgenic offspring. Although powerful, this technology...the Cas9 endonuclease that creates the DNA double-strand break (DSB) at the target site, and a donor oligonucleotide...
  24. Viral Vectors 101: Gamma-Retroviral Packaging Systems

    Type
    Blog Post
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    Aug. 6, 2024, 1:15 p.m.
    ...additional flexibility in pseudotyping.   Figure 1: Genome of a prototypical gamma-retrovirus.    ...an SV40 promoter, and Neomycin selection.   Table 1: Gamma-retrovirus glossary Plasmid Type Element...γ-Retroviral transfer plasmid encoding a transgene, sgRNA, or shRNA of interest: The transgene sequence is...packaging and transduction are not encoded by the transfer plasmid, but instead are provided in trans by other...integrated into the host genome upon viral transduction. Most transfer plasmids available from Addgene were ...packaging, and transfer) are supplied by three types of plasmids, which are cotransfected into the 293T...cell line. γ-Retroviruses can transduce target cells, but the transduced target cell does not produce ...
  25. All in a Twist: dsRNA

    Type
    Blog Post
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    Feb. 2, 2023, 2:15 p.m.
    .... Fig. 1: Exogenous sources of dsRNA Cellular sources of dsRNA Transposable elements such ... 286-301 (2022). doi.org/10.1038/s41580-021-00430-1 More resources on the Addgene blog CRISPR 101: RNA...Transcription, translation, and prone to degradation – those are the words that describe RNA! Double ...in our cells in the single strand form, but double stranded RNA and RNA:DNA hybrid molecules both exist...arise from a ssRNA engaging one strand of a dsDNA molecule; the 3-stranded structure produced is known as...intermediates in the process. These transcription-coupled R-loops are transient in nature and will dissociate...dissociate when gene transcription is complete. However, transcription of thousands of genes is constantly occurring...
  26. Targeted Mutagenesis with EvolvR

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    Blog Post
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    Feb. 21, 2019, 1:15 p.m.
    ...endonuclease activity degrades the displaced strand.   Figure 1: EvolvR consists of an error-prone polymerase...mutation rate and extend the editing window of EvolvR: (1) two additional mutations to PolI3M generated PolI5M...lineage tracing cells that do not tolerate double-strand DNA breaks by introducing a unique tag into a genomic...
  27. Plasmids 101: The protein expression toolbox

    Type
    Blog Post
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    Feb. 16, 2023, 7:05 p.m.
    ...blog! Fig. 1 CRISPR activator and repressor mechanisms Want to transiently induce expression..., they can deplete endogenous protein levels for 1-3 days. There’s also a CRISPR for that! CRISPR interference...small, double stranded RNA molecules that degrade target mRNA transcripts. When transiently introduced, ...fused to a transcriptional repressor to your gene of interest. It will inhibit transcription instead of...of introducing a DNA double strand break. Transient introduction of CRISPRi or inducible expression (more...LoxP vector.   Turn it off for a little while Transient suppression of expression is faster and easier...expression. Addgene has a dCas9 KRAB plasmid for transient knockouts!  Turn it on Turn it on forever Permanently...
  28. Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

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    Blog Post
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    Sept. 26, 2024, 1:15 p.m.
    ...effective with as little as 3 ng of DNA!) Figure 1: Schematic of SLIC. To start the SLIC cloning process...Methods Mol Biol. 2012;852:51-9. doi: 10.1007/978-1-61779-564-0_5. PubMed. JBEI-J5’s guide to SLIC Resources...through transformation, so the cloned vector must be ligated. But in LIC, longer single-stranded overhangs...T4 polymerase (left branch); by annealing two separate PCR products (middle branch); or by incomplete ...step to retain products with single-stranded overhangs (right branch). The mixed PCR and incomplete PCR...overlapping regions of single-stranded DNA. But as long as the double-stranded region is long enough to hold...nicks easily repairable by the bacteria after transformation. LIC is a reliable cloning method, but it is...
  29. FlipGFP, a novel fluorescence protease reporter to study apoptosis

    Type
    Blog Post
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    May 21, 2019, 12:10 p.m.
    ...contains 11 β-strands and one central ɑ-helix that can be split into 3 parts: (1) β-strands 1-9 (β1-9) and...and the one ɑ-helix, (2) β-strand 10 (β10), and (3) β-strand 11 (β11). Each part contains components and...developed, such as the Forster resonance energy transfer (FRET)-based executioner caspase reporters. In...need a protease reporter that has a large dynamic range and high brightness. The Shu Lab from UCSF designed...However, the lab wanted to increase the fluorescence range to better study the spatio-temporal dynamics of ...GFP (GFP1-9 and GFP10-11) by flipping a single β-strand of GFP, hence the name FlipGFP. FlipGFP increases...from β1-9 they will rapidly recombine with β1-9 strands nearby resulting in fluorescence within tens of...
  30. Viral Vectors 101: Viral Applications

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    Blog Post
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    April 13, 2023, 1:15 p.m.
    ...several advantages over traditional vaccines, including 1) cellular responses in addition to antibody response...and immunogenicity of attenuated, nef-deleted HIV-1 strains in vivo. Retrovirology. 2007 Sep 23;4:66. ...attractive due to the ease of transduction in difficult-to-transfect cell types ranging from primary to embryonic... traditional transfection and random genomic integration of a plasmid. Viral transduction is also generally...integrated into the genome of the target cell or transiently expressed. It’s important to note that for all...vector can express your construct in target cells transiently or constitutively depending on the virus used...generally less toxic to cells than transfections, preserving cell viability. Library delivery Screening...
  31. Viral Vectors 101: Biosensors

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    Blog Post
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    July 6, 2023, 1 p.m.
    ...Introduction to Biosensors. Essays Biochem. 2016 Jun 30;60(1):1-8. DOI: 10.1042/EBC20150001 Leopold, A. V., Shcherbakova... engineered to detect a wide array of neurotransmitters ranging from dopamine to glutamate, all the way... or animal system. This ‘thing’ could be a neurotransmitter, such as norepinephrine, or an electrolyte...last component of the biosensor is the signal transducer which will quantitatively give a readout for ...bio-recognition event is (fluorescence, pH, etc.). The transducer will generate a numerical value which represents...activated GFP expression (receptor), then the transducer could be a microscope, plate reader, or any other...—keep reading to find out how!   Neurotransmitters Many canonical fluorescent as well as FRET-based...
  32. Hot Plasmids - May 2022

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    Blog Post
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    May 26, 2022, 1:15 p.m.
    ... et al., 2021).    Fig. 1: Schematic showing the workflow for the BLITZ system. Image from Xiong...Biol. 2022. https://doi.org/10.1038/s41556-021-00836-1 Visual barcodes for live cell clonal multiplexing...use of pre-existing transgenic lines, termed BLITZ (Biotin Labeling In Transgenic Zebrafish) (Xiong, et...“single-use” transgenic lines. Instead, researchers can generate and maintain transgenic lines that can...protein by a specific transcription response element (TRE) or kinase translocation reporters (KTRs). Find...interest and, for in vivo work, generate a dedicated transgenic line. Using zebrafish as a model organism, the...protein can then be co-expressed in essentially any transgenic zebrafish line expressing a GFP-tagged version...
  33. Sweating the Small Stuff: Details in the Lab

    Type
    Blog Post
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    Feb. 8, 2022, 2:15 p.m.
    ... to focus, which was extremely effective. Others ran experiments at odd hours, used more secluded workspaces...consistently and accurately run a protocol.   Figure 1: Evolution of a Western blot protocol, from a full...
  34. OCaMP: A New Calcium Indicator for Neural Imaging

    Type
    Blog Post
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    Aug. 19, 2025, 1:15 p.m.
    ...Figure 1: Excitation (dotted) and emission (solid) spectra for various teal, green, yellow, orange, and ...spanning nearly the entire visible spectrum (Figure 1). The GCaMP series (e.g., jGCaMP8) remains the gold...fluorophores. The spectra for mOrange2 (scaffold of OCaMP) are shaded in orange. Data from FPbase.org. ...complement these tools, we recently developed a new orange calcium indicator, OCaMP (Aggarwal et al., 2025...2025), based on the fluorescent protein mOrange2 (Figure 2). Figure 2: OCaMP structure and mechanism...CC-BY-NC-ND 4.0 International license. Why orange? OCaMP’s emission peak at 565 nm fits neatly between...for two-photon imaging with newer lasers in the range of 1000–1080 nm. These industrial fiber lasers are...
  35. A Practical Approach to Choosing the B(right)est Fluorescent Protein

    Type
    Blog Post
    Published
    June 1, 2017, 2:30 p.m.
    ... and, if you do it right, can be (part of) figure 1 of your next paper or thesis. This series of posts...An explanation of the assay is depicted in figure 1. The strict correlation between the fluorescent protein...brightness When a dish of mammalian cells is transiently transfected with a plasmid containing a fluorescent...fluorescent proteins, e.g. see Chudakov et al (2010), Cranfill et al (2016) or Thorn (2017). However, the theoretical...developed in our lab uses a reference protein that is translated from the same open-reading frame and separated...reference protein) under identical conditions, a ranking of the practical brightness can be made (Goedhart...repeating this with another fluorescent protein, a ranking of practical brightness can be made. This strategy...
  36. Back to Bacteria: CRISPR gRNA Multiplexing Using tRNAs

    Type
    Blog Post
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    June 2, 2015, 6:06 p.m.
    ...modified slightly with either a 1-7 base poly(U)tail in tRNA-gRNA constructs or a 1-4 base tail from the second...tRNAs contain internal transcriptional elements that enhance pol III transcription, potentially increasing...base Csy4 recognition sites from a polycistronic transcript. Although this method shortens construct size...coexpression of Csy4 with Cas9 and the polycistronic transcript. As nucleases may have off-target effects and...matching traditional gRNA-expressing constructs, transcript levels of PTG1 and PTG2 were 3 and 31 times higher... of polycistronic gene organization, and its translation to CRISPR/Cas9 shows great potential for multiplexed...
  37. Troubleshooting Your Plasmid Cloning Experiment

    Type
    Blog Post
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    Sept. 24, 2019, 12:55 p.m.
    ...are working with is correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...the specific reaction you are running. Transformation Transform a few microliters of your ligation reaction...The PCR could fail to produce a product, the transformation may not result in any cells, or all the colonies...fragments If you don’t get any positive clone, try transforming the ligation mix in different bacterial strains...
  38. Deep Dive: Statistical Tests (Comparisons)

    Type
    Blog Post
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    March 7, 2023, 2:15 p.m.
    ...method entails arranging all calculated p values from smallest to largest and ranking them (1 being the smallest...error, variance, data ranges. how closely data points fall to each other (see Fig. 1). Alpha values –...statistical test assumes your data follows. Fig. 1: Examples of comparable groups with and without equal...significance by finding the largest p value where its rank divided by the total number of comparisons performed...– Tukey’s test, Newman-Keuls, Duncan’s multiple range test, and several others. Each test has different...negatives, moderate false positives. Duncan’s multiple range test – Most powerful. Low false negatives, high ...): 126-130 (2012). 10.1097/SMJ.0b013e3182498ad5 Curran-Everett, Douglas. Explorations in statistics: statistical...
  39. Choosing the B(right)est Fluorescent Protein: Aggregation Tendency

    Type
    Blog Post
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    June 15, 2017, 2:30 p.m.
    ...protein of interest will be problem-free. In figure 1 we provide an example of the OSER assay with a number...can be shared by publishing it as (part of) figure 1, uploading it as a preprint, or documenting it in ...not by Cranfill et al (2016)). Moreover, good performance in the OSER assay is not a guarantee that using...fluorescent proteins when protein tagging is the goal (Cranfill et al, 2016; Constantini et al, 2012). The localization...
  40. Suggest A Plasmid You'd Like to Find at Addgene

    Type
    Blog Post
    Published
    March 16, 2016, 5:30 p.m.
    ...to reach out to potential depositors in two ways: 1. Fill out our "Suggest a Plasmid" form Fill out the...where we can find the plasmid. While we cannot guarantee that everyone we reach out to will end up depositing...
  41. Predicting Adverse Reactions to Monoclonal Antibody Drugs

    Type
    Blog Post
    Published
    Feb. 13, 2024, 2:15 p.m.
    ...immunogenicity score (Mattei et.al., 2022).    Figure 1: Generation of immunogenicity scores by predictive...antibodies for the treatment of diseases. J Biomed Sci 27, 1 (2020). https://doi.org/10.1186/s12929-019-0592-z  ...ADAs can inactivate the drug and increase drug clearance, lowering its efficacy and increasing the risk...
  42. Antibodies 101: Stripping and Reprobing Western Blots

    Type
    Blog Post
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    Aug. 1, 2024, 1:15 p.m.
    ...Glycine-HCl 25–200 mM SDS 0.1%–1% (w/v) Tween 20 (optional) 1% (v/v) HCl Adjust to pH 2.0... your gel and membrane transfer as normal. Immediately after transfer, dry the membrane to maximize protein...you from having to run a whole new gel and membrane transfer. Let’s get started! Before you begin In the...manufacturer’s instructions for the membrane you are using. Dry nitrocellulose membranes are brittle, so proceed ... as normal! Stripping your membrane Alright, so you’ve dried your membrane and returned it to buffer, ...’ll want to: Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...a fume hood. Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...
  43. Overcoming the AAV Size Limitation for CRISPR Delivery

    Type
    Blog Post
    Published
    Sept. 16, 2020, 1 p.m.
    ...874 . https://doi.org/10.1038/nmeth.3993  Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu.../doi.org/10.1038/s41586-019-0908-x  Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani...949. https://doi.org/10.1016/j.cell.2014.02.001  Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...indel formation and phenotypic changes as early as 1 week post-injection. Livers from these mice were histologically...gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of ...prime editors. While using AAV to deliver large transgenes might seem daunting, it’s actually a challenge...split AAVs. In general split AAVs break a large transgene in two pieces and package each piece into an individual...
  44. New Tool for Lineage Tracing: The ClonTracer Library

    Type
    Blog Post
    Published
    Sept. 22, 2015, 2:30 p.m.
    ... of two outcomes (see schematic depiction below): 1. Resistant cells among the separate replicates would...development of combination therapies.         References 1. Hyo-eun, C. Bhang, et al. "Studying clonal dynamics...library was developed when Carrie was a post-doc in Frank Stegmeier’s lab in Novartis Oncology.  Bhang did...lentiviral vectors with roughly 73 million semi-random, 30 bp DNA barcodes that are integrated into target...
  45. CRISPR 101: Cas9 vs. The Other Cas(s)

    Type
    Blog Post
    Published
    Dec. 8, 2022, 2:15 p.m.
    ...none. Best for: targeting single stranded RNA.   Table 1: Fast facts on Cas family proteins...The main differences between Cas7-11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity.... it will proceed to generate a blunt DNA double strand break. Cas9 has been studied for genome engineering...an expanding number of others. Best for: a wide range of DNA edits.   Cas3 – The shredder What sets Cas3...locates its target sequence and initiates single stranded DNA degradation. Cas3 DNA digestion can proceed...convenient for targeting experiments requiring single strand DNA generation, such as homology directed-repair...therapeutic delivery.   Cas14 – Mini, but single strand mighty Unlike many other Cas enzymes, Cas14 targets...
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