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  1. How to Deposit Your Plasmids with Addgene

    Type
    Blog Post
    ...If your plasmid can be classified in multiple categories, please check all of the appropriate categories... a collection that is greater than 75 plasmids, please let us know by contacting [email protected]. ...information directly into the file. Email the completed spreadsheet to [email protected] along with ...plasmid can be added on subsequent pages. Type – please choose from one of the following descriptions for...used to knockdown expression of mRNAs with complementarity to the shRNA sequence. Empty backbone A plasmid...through cloning. *Note — If you will be depositing multiple plasmids and would prefer to send us your plasmid...e-mail us at  [email protected] Special cases Please contact us at [email protected] or +1 (617)-ADDGENE...

  2. Deep Dive: qPCR

    Type
    Blog Post
    ...across the board. If the samples or sample conditions change, the multiplexed protocol may have to be ... If there are multiple amplicons, or primer dimers forming, the curve will show multiple peaks.    ...optimizing for LightCycler machines.) Multiplexing It is possible to multiplex qPCR when using fluorescent probe-based...individually run sample when developing the protocol and optimized until the multiplexed and individual ... With the right conditions and parameters, some people find it can detect even smaller differences, and... can be the more optimal choices at times. For example, chemistries that rely on hairpin design of probes...the Lightcycler while being relatively easy to multiplex (Thellwell, 2000). See Table 1 for a comparison...

  3. Transferable Skills Guide: External Collaborations

    Type
    Blog Post
    ... worked with people outside the department to accomplish a variety of goals. For example, I worked with...experts that complement your (scientific) skill set and is therefore very useful for complex projects. It...met interesting people at conferences and you have successfully reached out to people outside your organization...Meeting people outside your organization just to have a quick chat can be rather tricky (think people based...Addgene I work with people from various organizations in science and beyond. For example, I help organize...collaborations are becoming increasingly important as more complex approaches to scientific questions become popular...mutual benefit.  Our partnership with SnapGene, for example, allows us to display high-quality maps and annotations...

  4. Hot Plasmids: Summer 2024

    Type
    Blog Post
    ... can be completely denaturated (left) or complexes may dissociate  (right). B) Protein samples protected...protect fragile samples during cryo-EM grid plunge freezing By Rachel Leeson Cryo-EM samples can be damaged...and structure determination LEA proteins protect samples during cryo-EM grid freezing Engineered and evolved...antibodies for marking astrocytes MagIC-Cryo-EM for sample enrichment and structure determination By Mike ...for low-abundance targets or natively isolated complexes. To address these limitations, the Funabiki Lab...enrichment streamlines the purification and minimizes sample loss while also supporting imaging, making it easy...cryo-EM grid.     Figure 1: MagIC-cryo-EM for sample enrichment and structure determination. A) Schematic...

  5. Antibodies 101: Immunoprecipitation

    Type
    Blog Post
    ...cells or tissues contain a complex mix of proteins seen as green squares, purple circles and blue triangles... adjusted depending on the sample. It is important to remember that sample lysates will also contain proteases...capture antibody. This will deplete the nonspecific proteins from the sample prior to beginning the IP....proteins are completely removed. Wash steps should be performed even with a pre-cleared sample.  Eluting ...parallel with both a sample known to express the protein of interest, such as a sample transiently transfected...immunoglobulins, to isolate a specific protein out of a complex mix. Using this technique, users can look for the...the antibody or antibody-target protein immune complexes, wash away unbound protein, elute and measure ...

  6. Troubleshooting and Optimizing a Western Blot

    Type
    Blog Post
    ...your samples. There are a few issues that can arise in this step: protein degradation, incomplete lysing...time.   Samples can run into empty neighboring lanes, so load your gel to ensure your samples and reference...wells. Most people will simply load an extra ladder or technical replicate (ie, leftover sample) to fill ...proteins going completely through the membrane or larger proteins not transferring completely onto the membrane...your protein gradient, compare your diluted samples. If multiple dilutions are showing the same signal (Figure...amount of protein/sample loaded Incubate primary antibody at 4 °C overnight Incomplete washing Ensuring...gradients. Protein gradients use dilutions of your sample or protein of interest to understand the dynamic...

  7. Antibodies 101: Flow Compensation

    Type
    Blog Post
    ...experimental samples. Let's look at an experimental example in Figure 2. Figure 2A shows the sample prior to...Figure 2: Compensation example based on experimental sample. Flow plots showing a sample before (A) and after...channel to another. When you label your samples with multiple antibodies, the fluorescent probes on the...Compensation example with single color control. Flow plots showing an APC single-color control sample before... your experimental samples In order to do compensation on your experimental samples, you will first need...apply to your experimental samples as they did with your single control samples: if the population needs...overlap in spectra is often called "spillover." For example, if your flow cytometer reads out the fluorophores...

  8. Getting to Know Expansion Microscopy

    Type
    Blog Post
    ...we just made the sample bigger? In ExM, and its various derivatives, biological samples are enmeshed in...label) your sample (i.e. cultured cells or tissues).  Step 2: attach anchors to your sample that will link...times, and the physical properties of the sample itself. For example, cultured cells are typically easier ...structure. For example, cytoskeletal structures that form continuous lines in pre-expanded samples can sometimes... popularity with an abundance of protocols and examples of applications from across disciplines now available...the number of specific targets you can label in a sample. Super-resolution microscopy (SRM), a family of... its original size. Depending on the protocol, samples can be expanded to ~4-10x linearly! This expansion...

  9. Deep Dive: Fixing and Permeabilizing for Immunofluorescence

    Type
    Blog Post
    ... permeabilize a sample. But you can also use organic solvents after fixing your sample with a cross-linker...during each step. Now that you know the basic principles of IF, we’ll take a slightly deeper dive into...protocols. Here we’ll start with just the first couple of steps - mainly, fixing and permeabilizing. ...the point?  Fixation allows you to freeze your samples in a given state or point in time. You want to ...processes while maintaining the structure of your samples, the relationships between cellular components,... molecules and it is great for preserving your samples and maintaining cellular and tissue morphology....cross-linker for IF. However, you might come across a couple other terms that can cause some confusion. Paraformaldehyde...

  10. Seven Tips for Using LinkedIn as a Scientist

    Type
    Blog Post
    ...connections This is a site for meeting people. Use the settings to let people see who you are when you look at...has spam profiles and trolls. People don’t like to connect with complete strangers as it borders on my... a LinkedIn profile is absolutely necessary. If people can’t find you on LinkedIn, you will lose opportunities.... When I am speaking at a new institution, for example, I will often see a whole bunch of notifications... (unless they’ve chosen to hide it). While most people use LinkedIn messaging, not everyone likes to. ...opportunity to start things out right. Also, many people on LinkedIn don’t appreciate random requests without...results in a generic invitation. 3) Connect with people you have really talked to The best time is within...

  11. AAV Titers: Where Do They Come From And What Do They Mean?

    Type
    Blog Post
    ...I’ll list the examples and you try to guess the category: ELISA...qPCR...Digital droplet PCR...DNA dot...each individual sample, this will reduce convenience and comparability because each sample with be quantified... low-concentration samples can also behave differently in PCR, even if both samples fall within the linear...reference sample of known titer, which is at a higher titer than the AAV RSM. By using these samples in all... titer values of new AAV samples. We also typically quantify our AAV sample by two qPCR assays and compare... viral vector preparation protocol, and we are pleased to report that over 95% of viral capsids in representative...Systems, Harvard University.    Which titer do people usually report? Addgene and other viral vector ...

  12. PCR Applications for Copy Number Variation Assays

    Type
    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...

  13. 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 ...

  14. Antibodies 101: The Basics of Western Blotting

    Type
    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...

  15. 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...

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

    Type
    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...

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

    Type
    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...

  18. 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...

  19. 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...

  20. 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...

  21. 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...

  22. 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...

  23. 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...

  24. Antibodies 101: Validation

    Type
    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...

  25. Transferable Skills Guide: Conflict Resolution

    Type
    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...

  26. 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...

  27. 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...

  28. Antibodies 101: Choosing the Right Isotype

    Type
    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...

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

    Type
    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...

  30. Antibodies 101: Introduction to Gating in Flow Cytometry

    Type
    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...

  31. 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...

  32. Networking Follow-up: I Met a Person. Now What?

    Type
    Blog Post
    ...important” or senior people. Focus on building relationships with peers and people around you naturally...run. Don’t worry about the people that don’t respond; focus on the nice people who do respond. Don’t call...connecting with people you don’t like or who do not show interest in you. Network with the people that you want... it’s about building genuine relationships with people you care about knowing. How do you follow up with...the encounter. As a side note, my guess is that people will not be exchanging business cards for awhile... you have something to ask or say. It can be as simple as “it was nice meeting you.” Include follow-up...who helped you succeed in any way. Don’t believe people who tell you that thank you isn’t necessary. I ...

  33. Pushing the Limits of DNA Assembly

    Type
    Blog Post
    ...expediently join multiple synthetic fragments, create multiple mutations in one or multiple fragments, and...joining multiple fragments of DNA to create a synthetically designed DNA sequence. There are multiple methods...becomes as simple as mixing fragments together with linearized vector for seamless joining of multiple mutations...standardized constructs and tools for the rational and simple shuffling of DNA regions. Golden Gate Assembly ...Assembly Tool which is freely available online. Its simple interface can guide you through both experimental...Assembly Mix include:  Generation of constructs with multiple point mutations Cloning sgRNAs Production of Yeast...Expression Cassettes Generating constructs with multiple point mutations NEBuilder HiFi can simplify multi-position...

  34. Antibody Validation for Flow Cytometry

    Type
    Blog Post
    ...and so it may be necessary to try multiple techniques. For example, we have found that some antibodies...detecting the target-of-interest in your assay and sample-of-interest. The approaches available broadly map...determining the selectivity of antibodies for other sample types and for non-surface (intracellular) antigens...protocol used and the abundance of the epitope in the sample, relative to cross-reactive antigens – that is,...approaches used will be different depending on the sample and target type. To start the validation process...antibody validation, and it is recommended to screen multiple candidate antibodies when possible. If cost is...the target at a robust level, similar to that in samples of interest. If you are working with cells that...

  35. Developing Lab Management Software for Biology

    Type
    Blog Post
    ...success or failure: In our second example we’ve added some simple but critical logic. Some steps fail... a new tube. In the first example we’ve used inheritance to tightly couple the contents of a tube with...appropriate glycerol stock within our freezers Pick the sample into the appropriate stab culture Grow the stab...proper location To keep ourselves from mixing up samples and to ensure that everything is grown successfully...needs to handle these cases. The following code examples show how software can be structured to allow for... for the messiness of biology. These examples are intentionally trivial, but they're here to help tell...tell this story. In this first example to the right (# this will make you sad),  someone has put in a request...

  36. Split Fluorescent Proteins for Studying Protein-Protein Interactions

    Type
    Blog Post
    ...compatible with multiple FP(11) fragments, even from a different base FP (for example, EBFP2(1-10) can...generally known as Bimolecular Fluorescence Complementation (BiFC). It can provide a qualitative or quantitative... or even single-particle tracking of the bound complex. There are so many FPs available, with many ways...because they are used with an already-available complementary fragment. Next, let's look at a round-up of...EBFP2(1-10) and Capri(1-10) for use with GFP(11) Multiplexed labeling of cellular proteins with split fluorescent...Cerulean Cerulean(1-10) for use with GFP(11) Multiplexed labeling of cellular proteins with split fluorescent...protein fragments for bimolecular fluorescence complementation analysis under physiological conditions. Shyu...

  37. Truncated gRNAs for Regulating Gene Expression

    Type
    Blog Post
    ...DNA strands complementary and non-complementary to the gRNA, respectively. When complexed with a truncated...full-length gRNA (20nt complementarity to target site) or truncated gRNA (≤15nt complementarity to target site...with which it is complexed rather than the protein itself. When native Cas9 is complexed with a gRNA that...transcriptional activator to the Cas9-gRNA complex. When this complex is directed to a target upstream of the...bioengineering, as it has facilitated the development of a simple and scalable means of making targeted genetic edits... directed to virtually any genetic locus when complexed with an appropriately designed small RNA, or guide...conventionally contains a 20-nucleotide sequence that is complementary to the target site, or protospacer, in the genome...

  38. A Guide to Starting Your Own Journal Club

    Type
    Blog Post
    ...group and topic. For example, it may be useful to adapt the second version (multiple presenter, figure by...time that accommodates more people. The feedback could be solicited via multiple avenues including casual...in journal clubs. They also have likely had the pleasure of attending both productive and not so productive...and maintain. It became clear that while several people across teams had various levels of experience with...decisions to make. How often will you hold it? How many people do you expect to attend? What is the expectation...ensure success. One of the most important is letting people know it’s happening. Aim for 3-4 weeks advance ...advance notice for the first meeting so that people can set aside time in their schedules and read the paper...

  39. INTEGRATE: Bacterial Genome Engineering Using CRISPR-Transposons

    Type
    Blog Post
    ...by expressing multiple crRNAs in parallel (Fig. 3), multiplexed insertions into multiple distinct targets... The requirement for multiple Cas proteins to form the Cascade targeting complex is a hallmark of Type...nuclease activity. Instead, it forms a novel co-complex with the transposition protein TniQ for targeting... by the system. Guided by the crRNA, QCascade complexes survey the genome and bind target DNA sequences...pairing. Separately, TnsABC forms a transposase complex with the mini-transposon, excises it from the donor... report of VchINT, three separate plasmids and multiple promoters were used to express the necessary components...in single-plasmid INTEGRATE (pSPIN), which offer simple transformations for smaller cargos, and the ability...

  40. Summer SciComm Series: Modes of Communication

    Type
    Blog Post
    ... pros of multiple channels/balance out cons. Flexible. Can allow people to learn in multiple ways from... means. Easy to combine multiple types of media. High barrier to entry (people have to enter a building... often unintentionally plays a big role in what people think of as "the best way" to communicate their...work just as well for everyone else?  In truth, people are incredibly varied in how they prefer to consume...However you choose to communicate your science, some people will love it and others will avoid it like the ...socioeconomic status and geographic location. For example, not all social media platforms are available or...probably didn't sound like a great idea to most people in 2005, but XKCD is pretty successful!  What resources...

  41. Hot Plasmids - May 2022

    Type
    Blog Post
    ...barcodes for live cell clonal multiplexing by: Rachel Leeson Multiplexed images in live cells remains ...own subclone, they were able to develop a 12-plex multiplex imaging system. Mixing the subclones within...effects of hi-multiplexing within in a cell, while potentially allowing for up to 20-plex experiments with...ever like to write about a recent plasmid deposit please sign up here. Here's what you'll find in this...knock-ins Visual barcodes for live cell clonal multiplexing Improving flexibility for in vivo proximity...Viviana Gradinaru Lab has previously developed the multiplexed version of the Cre-mediated AAV Targeted Evolution...mice and nonhuman primates (for AAV.CAP-B10, for example).  Given that the liver is an immunologically active...

  42. Advice for Starting a Biotech Company from a Grad Student turned Entrepreneur

    Type
    Blog Post
    ... into Sample6 as graduate students in Dr. Jim Collin’s Lab in 2009. From 2009 to today, Sample6 has changed...leadership, and grown to 25 members.  What is Sample6? Sample6 is an enrichment-free bacterial pathogen detection...their idea at various levels of complexity and abstraction and convince people with diverse backgrounds how...startup company. However, like many like-minded people, I quickly realize that a company requires more...current VP of Business Development & Operations of Sample6, for advice on how to take an idea developed during...etc. What project in the Collins lab inspired Sample6? The initial work focused on using bacteriophages... the focus of the application was changed, but Sample6 still leverages the core engineering and scientific...

  43. Binning Singletons: Tackling Conference Networking When You Don’t Know Anyone

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    Blog Post
    ...colleagues had moved on as well, so I knew very few people. I didn’t really fit in anywhere and everyone who...the meeting. I didn’t know anything about these people at the time, but I met some very accomplished scientists...with theirs.  It was at this dinner and the next couple of days of discussion with my dinner partners Denise...would provide a structure and support network for people attending large meetings. A key was to provide ...bin with a mentor who had attended the meeting multiple times before and would help the Singletons plan...help with the program on social media. Many of the people who participated as mentors or promoters were connected...vastly expand their networks as they naturally find people with which they have interests and experiences ...

  44. Transferable Skills Guide: Teamwork

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    Blog Post
    ...help of these people and others, we have updated our kit process substantially and have plenty of ideas for...group of people with distinct skills who fulfill a long-term function. When a select group of people are responsible...with other people, whether you want to acknowledge that or not. I just Googled the phrase “People need social... a leader can be successfully doled out to multiple people: someone to coordinate, someone to decide, ... have a specific purpose and finite goals. For example, when it came time to update how we make and distribute...function. Project team A Project Team is assembled to complete a series of tasks with precisely defined goals... in which case whoa! Mission accomplished! One example of a mission-based team in academia would be your...

  45. Intro to Our Summer SciComm Series!

    Type
    Blog Post
    ...the process of communicating science to laypeople.  Laypeople are simply non-experts in the field of science...would all be defined as laypeople when it comes to molecular biology.  Some lay people have a high amount ...other uses. Many laypeople would use “PCR test” to mean “a test that uses a biological sample to look for ...Table 1: Examples of defining your audience for science communication activities  Many people are concerned...language is aimed at a peer audience.  Note that lay people can and do consume scientific communication (it...use-case (definition) in a specific field.  For example, while “PCR test” has recently transitioned from...the possibilities are endless.    Activity Example audience  Talking about your career as a scientist...

  46. Plasmids 101: Secondary Nanobody Toolbox

    Type
    Blog Post
    ...), which allows for the following to occur: A) Multiple polyclonal secondary IgGs can bind to different...sites per antibody. C) IgG’s are big enough that multiple labels (green circles in figure 2 below) can be...incubated together without forming large oligomeric complexes. Antibodies are also too large to be expressed...Antibodies Bivalent Polyclonal Can be labeled with multiple dyes Commercially available Not recombinant...sacrifice Good tissue penetration Can be labeled with multiple dyes Greater resolution due to lower label displacement...strength to a standard IgG secondary antibody (Pleiner et al figures 4A-C). And since they have limited...for colocalization labeling of up to 3 targets (Pleiner et al figure 4D). Unlike large secondary antibodies...

  47. Viral Vectors 101: Optogenetic Tools

    Type
    Blog Post
    ...BioRender.com.   Opto-G-protein coupled receptors  G-protein coupled receptors (GPCR) regulate cellular...consider when choosing one.     Figure 1: Example of an experimental optogenetic setup. A light source...light-gated ion channels, optogenetic G-protein coupled receptors (OptoXRs), and photoswitches. Light-gated...within a narrower band of wavelengths to allow multiple tools, such as biosensors, to be used in tandem...cellular function through multiple intra-cellular signaling cascades. Optical methods for controlling GPCR...than traditional pharmacology techniques allow. Multiple photoswitches can be used in a single pathway ...awry in a disease state.      Figure 4: Example of an intracellular signaling pathway activated...

  48. Plasmids 101: Dimers and Multimers

    Type
    Blog Post
    ... type of multimer present in your sample. In these instances, please note that the supercoiled dimer band...plasmid monomer from a plasmid sample, or if no monomers exist in your sample, you could try linearizing ...known as multimers, and they are the result of multiple plasmid copies combining in a process known as...control when performing a diagnostic digest is a simple but effective method for detecting plasmid multimers...Bedbrook & Ausubel, 1976). In fact, if your plasmid sample has a mix of different-sized multimers, you may...agarose gel). If the presence of a multimer in your sample remains unclear after visualizing it on a gel, ...consistent with the size of a supercoiled plasmid dimer. Please note that it is common to see other plasmid confirmations...

  49. Addgene's Tips for Plasmid Quality Control

    Type
    Blog Post
    ... in-frame protein fusions, and multiple cloning sites. In the simplest case we have a standard backbone... that consistently receives around 200 new DNA samples every week. Here we will provide an inside look...own plasmids. Staying organized With hundreds of samples coming through our doors, the first step to ensuring...our deposit process, and then we carefully track samples through our lab using barcoded tubes and plates...growth instructions (temperature, strain, media supplements), suggested sequencing primers, or mammalian ...assemble your expected full plasmid sequences (examples include Benchling, Serial Cloner, and Snapgene...process. Always sequence verify Once the physical sample and its associated information are at Addgene, ...

  50. Working with Nuclear Receptors

    Type
    Blog Post
    ...coregulators exist as multiprotein complexes, a member of a single coactivator complex can serve as a rate-limiting...conduit to control the actions of the whole complex.  For example, the phosphorylation status of SRC-3 defines...afforded by coactivator complexes suggests that coactivators may be integrators of multiple cell signaling systems...datasets, for example, document the expression patterns of nuclear receptors across multiple different tissues...interact directly with NRs) exist in steady-state complexes with secondary coregulator (or co-coregulator)...co-coregulator) partners. The coregulator ‘core’ complex is composed of a tightly-bound invariant group of proteins... bound co-coregulators associate with the core complex in a dynamic, regulated manner. The fact that coregulators...
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