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  1. Visualizing Your Plasmid Request Data

    Type
    Blog Post
    ...country. This information can be of personal interest to the depositor, but it can also be valuable data ...to parse through, aggregation of this information can provide valuable insight to the most requested plasmid...launched, it will show my data by default. Depositors can download their CSV file from Addgene by following... code is available in the Github repository (you can find the link in the upper right hand corner of the...dashboard). Therefore, users with some knowledge of R can further customize the dashboard to their needs. I...uses genetically encoded fluorescent probes. You can follow him on twitter: @joachimgoedhart.    ...

  2. Newly Updated AAV Data Hub!

    Type
    Blog Post
    ...reports (which you can filter by serotype, species, or promoter) can save you significant time and money.... and we've updated our submission process so you can easily add your data, too!   Using the Data Hub...between BLA projection neurons and mPFC interneurons can be labelled with an AAV1 Cre virus combined with ...virus in the mPFC.  The AAV9 is so strong that you can even see dendritic spines!  Curious? Watch our AAV...'ve ever validated an Addgene or CLOVER AAV, you can (and dare we say, should!) submit your data to the...off, thanks to our handy-dandy "save" button. You can check out our step-by-step submission guide or email...

  3. Quick Guide to All Things Lentivirus

    Type
    Blog Post
    ...after transfection. This supernatant can be stored at 4 °C or can be directly added to your target cells...including human immunodeficiency virus type-1 (HIV-1), can infect both dividing and non-dividing cells. The ... This generation is safer than the first one and can be used routinely in a research laboratory. The risk... production. Third generation lentiviral vectors can be used for research and for clinical purposes, however...titer than with the 2nd generation. Knowing this you can scale up your production set-up and purify and concentrate...cells of interest.   Advantages Disadvantages Can carry large transgenes (>8 kb) Potential for generation...Replication-incompetent lentiviruses with human tropism can still infect the researcher and insert into her/his...

  4. 9 tips for a successful postdoctoral experience

    Type
    Blog Post
    ...mentors often can be found during conversations with senior investigators. Discussions can reveal mutual...taking this time, you can help ensure that you understand the methodology and can have confidence in the...: What is the most exciting story I can tell with my data?  Can I make better slides that more effectively...telling your story, you can find clearer ways to consolidate or present your data. You can create more detailed...Experimental successes can be infrequent and a hobby that provides instant gratification can help boost your ...search committees, and can lead to mutually beneficial collaborations. 7. You cannot get too much practice... fellowship, what are you signing up for and how can you do it well? Below, I briefly describe some tips...

  5. Troubleshooting Your Plasmid Cloning Experiment

    Type
    Blog Post
    ... many softwares out there than can help you at this stage and that can be used to simulate in silico cloning...There are several online tools that can help you with that, they can be open source (i.e. primer3) or provided...product development scientist at Addgene. Cloning can be quite an arduous process. The PCR could fail to... contain the correct plasmid. There’s a lot that can go wrong! With all the steps in the cloning process...scheme, you’re now ready to generate fragments. You can generate the parts of DNA that you want to assemble... template, particularly from a large genome, you can recheck your primer design or optimize your primers...Assembly of the fragments There are multiple ways you can assemble the different parts of a plasmid based on...

  6. Stabilized Bacterial Promoters: Constant Gene Expression at any Copy Number

    Type
    Blog Post
    ...temperature, and growth rate can all impact plasmid copy number in a cell. One can attempt to eliminate fluctuations...result, variable gene expression can make data hard to interpret or can interfere with the precision needed... time resulting in variable gene expression that can impact observed phenotypes. Factors such as the growth...chose a TALE protein as the repressor because TALEs can be programmed to bind tightly to arbitrary DNA sequences... slowly or quickly Growth media and growth stage can both affect gene copy number. Moreover, gene copy... gene expression. On the other hand, slow growth can also cause changes in plasmid copy number. During...genome or a plasmid. Thus, a stabilized promoter can allow a system to be moved from a plasmid to the ...

  7. Antibodies 101: Buffers, Storage, and Conjugates

    Type
    Blog Post
    ...manufacturer, lot number, and expiration date somewhere you can easily check it whenever you use the antibody. This...experiments.  Now that that’s out of the way, you can put your antibody in short term storage (think days...best option, in aliquots no smaller than 10uL. You can leave them at room temperature when you’re working..., a common antimicrobial agent added to buffers, can bind to amine groups present in some conjugates, ...noted on the product sheet. Thankfully, sodium azide can easily be removed from the buffer and then re-added...cases, thimerosal at a concentration of 0.01% w/v can be used as an alternative antimicrobial.  Conjugates...mostly use HRP, a chemiluminescent conjugate that can be activated to emit light with a simple kit, and...

  8. Plasmids 101: Sequence and Ligation Independent Cloning (SLIC)

    Type
    Blog Post
    ...ligated. But in LIC, longer single-stranded overhangs can hold a nicked plasmid together long enough to get...′ exonuclease activity. T4’s polymerase activity can be thwarted if no dNTPs are available to polymerize...type of dNTP in the reaction mix, the researcher can decide exactly how far the T4 exonuclease proceeds... create 10–12 base “chewed back” overhangs, they can be annealed, forming a circular product with four...homologous recombination intermediate to bacteria, so it can be transformed as-is into cells. The cells then get...itself is flexible. For one thing, those 5′ overhangs can be generated in several ways. One option, imprecise...be digested in the same tube. Alternatively, SLIC can also work directly on PCR products, with 5′ overhangs...

  9. Choosing a CRISPR Nuclease: Site Accessibility, Specificity, and Sensitivity

    Type
    Blog Post
    ...This can be determined by measuring the indel frequency at a target site. A specificity ratio can be measured...of genomic loci that can be targeted (Hu et al., 2018).  These altered variants can allow for targeting...accessibility is a measure of which genomic sequences can be targeted by the Cas9. This is determined by the...sequence is 3’-NGG. But Cas9s found in other species can recognize a variety of different PAM sequences. Researchers...accessibility. Cas9s with a broader PAM specificity can target more sites. Created with BioRender.com. ...assessment of the off-target activity of Cas9. This can occur when Cas9 cleaves a site that is not a perfect...genomic locus of interest. As these off-target effects can be genome-wide and, in some cases, difficult to determine...

  10. CRISPR Meets Synthetic Biology: A Conversation with MIT’s Christopher Voigt

    Type
    Blog Post
    ...Addgene: What can CRISPR do for synthetic biology? Voigt: The big promise is orthogonality. You can create an...CRISPR is so orthogonal and programmable that you can make a very large number of regulators. That basically...access that type of material - or do what biology can do - it’s good to be able to build regulation thats... is toxic. When cells carry CRISPR circuitry, it can slow their growth. Something has to be done about... because they are used to create any program you can imagine. The Apollo 11 mission circuitry that took... a fundamental unit. We did demonstrate that you can use the CRISPR chemistry for a NOR gate. But there...worked. I don’t have the solution. Our paper shows we can easily make orthogonal variation. The first one worked...

  11. Plasmids 101: Yeast Vectors

    Type
    Blog Post
    ...which can be easily maintained when grown on media containing the missing nutrient. Scientists can exploit...the marker gene) For use in: Can this be used in other yeast species? Can this be used for auxotrophic... maintenance within an E. coli cell, but vectors can be widely utilized across many different cell types...those of plants and animals. Unlike bacteria, yeast can post-translationally modify proteins yet they still... chromosome. As the name suggests, these vectors can replicate independently of the yeast chromosome; ...propagate per cell. The copy number of these vectors can also be controlled if specific regulatable elements... yes - Grow with 5-FOA. S. cerevisiae yes - This can complement ura4- S. pombe, but the complementation...

  12. Tips to Make the Most of a Scientific Conference

    Type
    Blog Post
    ... scientist whose work you are familiar with. You can tweak your elevator pitch slightly if you know a ...you have a poster or talk at the conference, you can end by asking them to come to your poster or talk...your agenda. Some conferences even have apps you can use to build a schedule. If there is a list of poster...this information on the poster) so your visitors can have a DOI to go to. During the conference Arrive...through the poster.” Have a notebook handy so you can jot down notes or questions from your poster viewers...media. Social media Before the conference Networking can be made easier through social media. Use social media...If you could not attend some of the sessions, you can see if others have live-tweeted as well. In addition...

  13. Making CRISPR Plasmids Using Fragmid

    Type
    Blog Post
    ...yourself with a common dilemma. Request the vectors you can find and use them as-is, saving time and effort but...cloning the vectors you actually want. But now you can use the recently developed third option: Fragmid....are the components. Using the handy website, users can simply select the components they want for each module...selection markers. Need fewer components? A spacer can be inserted instead, allowing everything to stay ... individual plasmids and as the Fragmid kit. You can also browse all Fragmid components on the Fragmid...restriction digest, and then plasmid sequencing if you can. To quote Doench, “Whole plasmid sequencing is just...recognition sequence. This means that each component can be designed with an overhang specific to the type...

  14. Fluorescent Proteins 101: Aptamer Fluorophores

    Type
    Blog Post
    ...ratios with such techniques can be problematic, however. Well designed aptamers can help reduce such background...temperatures, since proteins can denature while aptamers are more stable and can go through cycles of denaturation...bacteria). That range is possible because aptamers can either fold to incorporate their target molecule,...enzymes, such as: Design and selection for targets can be done largely in vitro; Easy and reproducible ...antibodies for diagnostics know how frustrating it can be to get a “bad batch”); Customizable sensitivity...aptamers glow The flexibility of aptamers means they can be used with or as fluorophores in several different...aptabeacons (or aptatags) were born. Aptabeacons can be created in many different ways, but the general...

  15. Viral Vectors 101: Producing Your rAAV

    Type
    Blog Post
    ...viral particles that can transduce cells, which can vary based on target cells and can be altered by freeze... where you can obtain excellent quality, high titer rAAV (including Addgene!), but you can also generate...due to their high density, which can be collected afterwards. You can then remove the density gradient...system, HEK293T cells, expressing Ad E1A and E1B, can be transfected with your transfer plasmid, carrying...titration by qPCR.   Pro tip! An optional media exchange can be performed 12–18 hours post-transfection to help...For certain in vitro and in vivo applications, you can consider using unpurified AAV from the supernatant...Purification To purify your rAAV from your supernatant, you can simply use polyethylene glycol (PEG). We add 25 mL...

  16. Deep Dive: qPCR

    Type
    Blog Post
    ...and analysis.  As a rule of thumb, you can assume that qPCR can accurately detect expression differences...differences, and it can even be used to detect a single copy of a transcript. But this sensitivity can sometimes...configurations can help you identify pipetting or evaporation errors. And if time allows, you can fit in a ...statistical significance (Taylor, 2017). With the right conditions and parameters, some people find it can detect... requires significant optimization to ensure the results are correct. Primers and probes can interfere...troublesome, multiplexing your reference gene set can save significant plate space and may be worth the initial...to measure relative quantification of mRNA, which can be used as a proxy measure for differences in protein...

  17. Antibodies 101: Avoiding the Mouse on Mouse Mess in IHC

    Type
    Blog Post
    ...in non-specific staining can be a challenge in the best of cases. But it can be particularly challenging...(Figure 2B) Some labs have found that you can significantly decrease background staining by mixing your...same levels of endogenous IgGs, so some researchers can get away with species on species staining with no...    Figure 1: Species on species staining can be a problem because the secondary antibody is not... However, directly conjugated primary antibodies can be expensive and may not provide enough signal to...1998). After washing away unbound fragments, you can then apply your primary and secondary antibodies ... Proper tissue processing and sample preparation can also help reduce the levels of endogenous IgGs in...

  18. Mentoring for Scientists: I Have a Mentor, What Now?

    Type
    Blog Post
    ... fun example and you can find it in our Mentoring for Scientists Guide.  You can use it to clarify and...step you can take is to set goals to work on together and track progress at every meeting. It can take many...impart. An undergraduate can mentor a high school student, a graduate student can mentor an undergraduate...Series. The entire series and additional resources can be downloaded in E-Book format at the end of this...setting to your mentoring relationships, so that you can reap rewards in the form of reaching career development...undergraduate, a postdoc can mentor a graduate student – you get the idea. There are many advantages to being a ... Schedule meetings or calls regularly and don’t cancel, so that you drive progress with regular accountability...

  19. Transferable Skills Guide: Creativity

    Type
    Blog Post
    ...scientist. In this blog, we’ll delve into strategies you can use to foster innovation. If you identify as a non-creative...yourself a block of time and a quiet space where you can address the issue head on. Prepare for this time ...environment where everyone has their ideas heard.  So how can we assure that all participants feel comfortable ...devil’s advocate is elected, others may feel they can share opposing opinions as well. I am sure we have...goal is to generate unique ideas. Recognize ideas can come any time. Meetings shouldn’t be the only place...ideas are not well received, understand that people can be resistant to change. Ask your peers or your superiors...fixated with the idea of making something the best it can be that it seems impossible to start at all. This...

  20. 10 Steps to a Perfect Science Talk

    Type
    Blog Post
    ...the best speaker you can be. Seek out opportunities to practice and over time you can master this important...solutions—public speaking is skill that any scientist can learn.  Any time you give a science talk, you are... added bonus to knowing your audience is that it can help you avoid making mistakes. I once made a joke...a narrative connecting cause and effect. Stories can and should be very simple. The goal is to get the...to include that material, but don’t force it. You can always relate to scientists by explaining how your... real audience Good, even great, speaking skills can be attained by almost anyone with enough practice...previous year. Usually fewer than 10% have done so. How can you expect to deliver a great public presentation...
Showing: 261 - 280 of 1075 results