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Showing: 161 - 180 of 1010 results

  1. PCR Applications for Copy Number Variation Assays

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    Blog Post
    ...irivbrro ddPCR  Droplet digital PCR (ddPCR) works by using oil droplets to partition a single sample into thousands... reference sample with two copies of the gene (Ma, 2014.) As with all qPCR assays, samples, standard curves...see image, left, of droplet immersion technique, courtesy of CAR15.) Each droplet contains a primer and...reference sequence present in the same amount in all samples (usually to a ubiquitously expressed, one-copy ...housekeeping’ gene), and quantified relative to a 'normal' sample with known copy number set as the expression baseline...since you’re looking at the amount of DNA in your sample relative to your normal control - you can set up...number (RQ value) matches the copy number of your sample. In that case, an RQ value of one (or, more realistically...

  2. Antibodies 101: Flow Cytometry Controls

    Type
    Blog Post
    ...controls An isotype sample is prepared very similarly to a FMO sample: you make a sample from a combined ...your samples. The single color controls should be used as a starting point, to preserve your sample, and...adjust the settings with experimental samples. Figure 1: Example flow plots for a single color control...experiment. To make an FMO sample, you take a subset of your experimental samples, combine it into one well...population. Figure 2: An FMO gating example. The FMO sample (A) can be used while setting up your gating...  Figure 3: Isotype flow plot example. The isotype sample (a) can be used to confirm that your ...controls will help you save on sample volume, so you can use most of your samples for flow analysis rather ...

  3. Antibodies 101: The Basics of Western Blotting

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    Blog Post
    ...size and density present in all samples. Uneven sample loading or incomplete protein transfer causes inconsistent...thiols, to cleave disulfide bonds. Load the samples, one sample per lane, onto the top of a resolving gel...simply the western blot, or western, is one of the simplest methods to detect the presence or absence of a...Figure 1: Overview of the Western blot process. The sample, a mixture of protein is separated in a gel, transferred...to detect a single, specific protein within the complex mix derived from cells or tissues. Westerns are...is the first step of a western. To prepare the samples for SDS-PAGE, measure the protein content and normalize...normalize to ensure equivalent loading. Denature the samples to their primary amino acid sequence by boiling...

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

    Type
    Blog Post
    ...data from multiple primer pairs, one can infer the predominant state of registers within a sample using a...alternative. The qPCR method can be performed on a sample of multiple registers. However, it is only semi-quantitative...states in a sample; instead, it will suggest the predominant state of registers in a sample based on a ...methods do not. How might one implement RSMs? In our study (1), we implement RSMs in E. coli with two plasmids... often times there are multiple register designs that may be used to implement the same gene regulation...needs. For more context, exposition, and detail, please refer to the paper. Why might one be interested... achieved by expression of other recombinases. Multiple orthogonal attB-attP pairs per recombinase may...

  5. Behind-the-scenes of the Isolation of the Thermostable IgnaviCas9 From a Yellowstone Hot Spring

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    Blog Post
    ...sequence this highly complex hot spring sample. The sample was separated into smaller samples of 5-10 cells ... Mork collecting samples at the hot spring. Hiking all day and processing samples by night was fun...leave your samples behind! Paul and Anastasia (Potanina) Neddersen characterized a few samples by 16S ...from these extreme environments. Gearing up for sample collection at the hot springs My husband, Paul ... and two deep cycle batteries for power so the samples could be kept frozen after collection. Figure...challenges: collecting and processing environmental samples Since the high temperatures and unstable ground...dangerous, we used telescoping poles to collect water samples from a distance. At each collection site, we took...

  6. Lab Automation at Addgene

    Type
    Blog Post
    ...pipette samples in and out of it, you can set up a procedure for it using the Biomek FxP. For example, we ... pretty simple. By clicking a few buttons you can quickly tell the Biomek FxP how much sample to pick ...easy to spread out your samples. This can be useful if you’re transferring samples from 96 wells plates ... from many bacterial samples at the same time as opposed to preparing each sample individually. Enter ...provide you with some tips on how to automate some simple processes in your own lab. Automating bacterial...warming up too much and dying.  Also, we added a couple of platforms that fit 48 well q- trays allowing...mobile pods - one pod can pick up 96 tips for easy sample transfer between 96 well plates. The second pod...

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

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    Blog Post
    ...high-quality isolated DNA samples of sufficient quantity for sequencing. This process is completed in a plate format...MiSeq that takes about two days to complete. After the run completes, we begin our assembly process. Again...holes are then filled with other samples in our queue such as viral samples for Viral Genome Sequencing (... libraries.  Viral samples are filled into the same process as our normal Complete Plasmid Sequencing ...unique barcodes per sample so we can assign sequencing reads to the correct sample. The PCR is then performed...yielding anywhere from two to six plates of 96 samples per week. This is where our new process will begin...begin. Partnering with seqWell, we are using the plexWell technology to easily and quickly create Illumina...

  8. Simplify Cloning with in vivo Assembly

    Type
    Blog Post
    ...the primer sequences. Multiple plasmids can be included in a single PCR, for example for subcloning genes...perform multiple recombination events, all linear fragments need to get into the same cell. For simple cloning... deletions, and point mutagenesis to complex assembly of multiple genes. Primer design for each type of...Combining multiple modifications A key feature of IVA cloning is the ability to combine multiple modifications... the multiply modified product. For example, by combining multiple primers, a C-terminal epitope tag can...GFP-tag (Figure 3). Achieving multiple modifications requires multiple recombination events to assemble...allow any cloning procedure to be performed using a simple two-step, 2 hr protocol prior to transformation...

  9. 27 Hot Plasmids from 2016

    Type
    Blog Post
    ...new assembly platform for generating both simple and complex DNA constructs Have you ever wished you ...it comes to simultaneously modifying multiple loci (multiplexing). Currently, the only genome engineering...revolutionized genome editing in multiple organisms thanks to its simple, easily modifiable RNA-guided targeting...S288c strains since failing to fully complement strains with multiple auxotrophies or using different auxotrophic... one third of all medications target G-protein coupled receptors (GPCRs) yet one third of the GPCRs in... and basic biologists alike often need to test multiple expression systems before they achieve the appropriate...have greatly simplified the process of generating multiple E. coli gene expression plasmids with the Cross-disciplinary...

  10. Typing CRISPR Systems

    Type
    Blog Post
    ... as Cascade (CRISPR-associated complex for antiviral defense) complexes, while Class 2 use single protein...tools using a Cascade complex is tricky, as you need to ensure every unit of the complex expresses at similar...present in the Cascade complex. Figure 2: Schematic of a Cascade complex (blue subunits) with a...primary effector complex but are defined by the accessory Cas proteins in the Cascade complex. Despite a tenuous... it can process multiple gRNAs under a single promoter, allowing for easy multiplexing. The double-stranded... of CRISPR technologies. Learning the general principles of this classification system will help you gain... your own research! Classification Structure Principles of CRISPR classification While CRISPR systems...

  11. Antibodies 101: Secondary Antibodies

    Type
    Blog Post
    ...approach is as follows: Prepare the sample. Incubate the sample with the primary (unconjugated) antibody... but it probably should be. In a multiplex assay - one with multiple proteins of interest - the secondary... simplifying multiplex assays.  A brief note Since the indirect method relies on multiple secondary antibodies...antibody instead of the primary antibody. Since multiple secondary antibodies can bind to a single primary...in blue; secondary antibodies are represented in purple; signaling molecules are represented in light green...stem of an antibody’s Y) is large enough to allow multiple secondary antibodies to bind to a primary antibody...remove any unbound or excess antibody. Incubate the sample again with a conjugated secondary antibody, which...

  12. Plasmids 101: Modular Cloning

    Type
    Blog Post
    ...biology are not so simple, and engineering complex cellular behaviors requires making complex manipulations...understanding MoClo. By a similar principle, TUs can be further complexed into multi-TU constructs, known...Level 1: Assembly of multiple Level 0 parts into TUs. Level 2: Assembly of multiple Level 1 TUs into multigene...restriction enzyme and a ligase are used to assemble multiple DNA fragments together in a one-tube reaction....predictable behaviors. While a gene is the most obvious example of an individual part, this category also includes... (TUs), which are functional units composed of multiple parts transcribed together. They can express one...begin with a promoter and end with a terminator. A simple TU could be comprised of just three parts: promoter...

  13. Antibodies 101: Validation

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    Blog Post
    ...bind to - particularly when you start using complex samples such as whole cell lysate or tissue sections...antibody signal in the retinal sample, but little to no signal in the liver sample, which you do (C), so your...signal in samples that have had the target protein expression experimentally increased. The simplest version...antibody works! Multiple (Independent) Antibodies Strategies These strategies use multiple, unique antibodies...require variable expression of your target across samples - it is hard to distinguish between specific, invariable...proteomic and transcriptomic datasets already exist.   Example: You want to use an antibody to examine the levels...there is a wealth of transcriptomic data from this sample type, so you use your bioinformatics skills (or...

  14. Transferable Skills Guide: Conflict Resolution

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    Blog Post
    ...conflict. Most people perceive any conflict as inherently bad, and in many cases it can be. If people or teams...one of your strengths. Empathizing with people, especially people with whom you strongly disagree, is a ...there to arbitrate for them. They were different people with some very different opinions, and some of ...’t just about resolving conflicts between other people, but resolving your own conflicts as well. What...strengthen. The right set of transferable skills, coupled with your technical skills, can open new career...”. A personality-based conflict occurs when two people cannot agree or get along with each other, regardless...left unchecked, this type of conflict can lead to people disagreeing with each other just for the sake of...

  15. Technical Design of a Western Blot

    Type
    Blog Post
    ...protein expression varies from sample to sample. To compare one sample to another, you’ll need to normalize...curve, your standard, and your sample(s), as you can only compare samples to a standard curve generated...published antibodies, check the manuscript and supplementary figures for validation data, or follow the references...western blot. Polyclonal antibodies, which bind to multiple sites on an antigen, are cheap and can help amplify...overnight or 1–2 hours at room temperature (RT). Most people choose to incubate their primary antibody overnight... likely higher background noise as well, and multiplexing is challenging and limited. Direct westerns ...conjugated to primary antibodies). Direct westerns are simpler and faster than indirect ones and can be used in...

  16. Transferable Skills Guide: Creativity

    Type
    Blog Post
    ...place people share ideas. Ideas had over lunch are just as valuable! In 2019, Addgene implemented a company-wide...funding in the world? What if you could hire more people? What would you do with unlimited time? Another...brainstorm session is to start with something completely unrelated to the issue and consider how it could...expert at Stanford conducted an experiment in which people were asked to create a device to help students ...avoiding groupthink, the tendency of a group of people to agree with the most prominent idea, and instead...comfortable sharing their ideas? And how do we give people the opportunity to speak out and prevent groupthink...you meet. This holds the dual purpose of giving people time to process the issue properly while also discouraging...

  17. Antibodies 101: Choosing the Right Isotype

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    Blog Post
    ...choosing antibodies   Complexity is a key factor Biological systems tend to be complex with a variety of ...Experiments tend to be equally complex with users trying to probe multiple targets in parallel. In these...antibody) approach?  Staining for multiple targets When staining multiple targets indirectly, you must strategically...If you are lucky in lab life, you will have a plethora of antibody options for your experiment (all well...Some secondary antibodies are broad and recognize multiple isotypes from a species, like a general anti-mouse...than a broadly  reactive secondary (Fig. 1). For example, an anti-mouse IgG2a secondary would be a better... rabbit versus mouse monoclonal antibodies for example. Rabbit monoclonal antibodies have higher affinity...

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

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    Blog Post
    ...flat-bottomed plate. The basic principle of an ELISA is that any antigen within a sample adheres to the well (either...wells with an experimental sample allows for detecting one protein from the sample. A direct ELISA has many...require scientists to detect proteins in their samples. One technique that relies heavily on antibodies...antibodies coating the wells to capture antigen from a sample, whereas antigen is bound directly to the wells...protein you are interested in detecting in your sample. When an ELISA kit is ordered, the wells are typically...already coated with the antibody. Then, you add your sample to the wells, and the antibodies bind to your protein...ELISA is best used to detect proteins for which multiple antibodies that recognize different epitopes are...

  19. Antibodies 101: Introduction to Gating in Flow Cytometry

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    Blog Post
    ...individual experiment. Practical example: lymphocytes in a mouse tumor sample You made it! So much about the...When using flow cytometry to analyze your samples, it is necessary to set up a sequence of gates to be...working with a heterogeneous cell population, for example from a processed piece of tissue, where cells are...variations in those factors. To understand the principles of gating, you’ll first need to learn a little...size, while SSC values depend on the structural complexity inside the cell or on its surface. Bear in mind...on the cell’s size, morphology, and structural complexity.   Height, Width, and Area Within FSC and...the area under the resulting curve. By default, a sample on the flow cytometer (before you set the first...

  20. Tips for Getting a Faculty Position

    Type
    Blog Post
    ...Perfect Science Talk." 4. The chalk talk, simple in principle The first question I usually get about chalk...relate to, that people would agree is important to understand. (1-2 sentences). For example: Distinguishing...you have any questions, comments or suggestions, please do not hesitate to email me at [email protected]’ve provided guidance and mentoring to several people that found my insights helpful. All went on to ... need a Nature/Cell/Science paper. You do need multiple first/corresponding author publications in quality...for feedback), possibly the Dean, and even some people in your field that still need to understand your...slide deck, markers, etc. Chalk or markers force people to be more succinct. Slide decks leads to the opposite...
Showing: 161 - 180 of 1010 results