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  1. Plasmids 101: The protein expression toolbox

    Type
    Blog Post
    ... a gene (cDNA) can be done by either plasmid or virus. A plasmid carrying your cDNA can be targeted for...single exon) and everything in between those sites can be excised by intentional introduction of the Cre...mRNA transcripts. When transiently introduced, they can deplete endogenous protein levels for 1-3 days. ...your choice cDNA. Both lentivirus and retrovirus can package cDNAs for genomic delivery and have many ...expression. There are several flavors of activators which can be used with this approach - read more about them...you! Many of the tools highlighted in this section can be used to turn expression on/off for short or long...tags Tagging relevant proteins with a degron tag can provide inducible protein degradation. These systems...

  2. Plasmids 101: Screens vs. Selections

    Type
    Blog Post
    ...condition that can apply enough pressure to select for variants of interest, though. A selection can take the...The process can range from a defined pathway or reaction to a complex event such as cancer or even aging...identify new players in a biological process. Players can be proteins, genes, enzymes, drugs, or anything else...that’s where the differences in these approaches can be found. As an example, say you are considering ..., you are not specifically looking at cells that can survive. Instead, all manipulations are assessed,... drug-containing media in which only cells which can tolerate the drug survive and everything else dies... to look at an outcome. Your scientific question can help determine which is right for you. Screen variations...

  3. Plasmids 101: Cre-lox

    Type
    Blog Post
    ...going to take a look at a very interesting tool that can be used for creating (excuse the pun) specific, targeted...Cre-lox? The Cre-lox system is a technology that can be used to induce site-specific recombination events...recombination event between two loxP sites, which can be located either on the same or on separate pieces...resulting in the reciprocal crossover event. This event can have three general outcomes based on the location...translocation event is generated at the loxP sites. How can I use Cre-lox? The Cre/lox system is a well-established...selection process. By floxing the selection marker, Cre can be used to easily perform this eviction. Regulated...with tamoxifen or doxycycline), the Cre recombinase can be expressed only in specified cells or at specified...

  4. Plasmids 101: The Wide World of Natural Plasmids

    Type
    Blog Post
    ...factors can also be integrated into the bacterial chromosome, but when encoded on plasmids, they can promote...around us! A plasmid is a small piece of DNA that can replicate independently from its host’s chromosome...we think of plasmids, both Archaea and Eukaryota can also harbor these circular DNA passengers. Most natural...keep them around and even help them spread. They can be inherited by daughter cells during cellular reproduction...reproduction, similar to the host’s chromosome, or they can spread between hosts through horizontal (or “lateral...lateral”) gene transfer. Horizontal gene transfer can occur naturally through transformation (cellular uptake...lab specimens — there is evidence that lightning can also promote gene transfer in soil bacteria! (Demanèche...

  5. Plasmids 101: Gateway Cloning

    Type
    Blog Post
    ...traditional restriction enzyme cloning. Instead, you can choose a molecular cloning technique that will work...the appropriate entry and destination vectors, one can use Gateway to clone a gene of interest into a variety...Under certain conditions, the attL and attR sites can recombine, leading to the excision of the phage from...modified versions of the att sites so that scientists can easily clone in their desired DNA sequences. Gateway...is toxic to the cell, Gateway cloning efficiency can reach >99%.   STEP 3: Express your Gene of Interest... via sequencing or restriction digest! Then, you can transform or transfect the cells that you want to...entry clone with your DNA sequence of interest, you can move this DNA fragment across any expression system...

  6. Antibodies 101: Immunoprecipitation

    Type
    Blog Post
    ...out of a complex mix. Using this technique, users can look for the presence or absence of a protein, determine...sites free to capture the target protein. The beads can be collected by centrifugation (agarose beads) or...recombinant protein, Protein A/G, was developed, which can be used for most antibodies compatible with either...for binding sites. In cases like these, antibodies can be directly conjugated to agarose or magnetic beads... not require centrifugation between washes which can cause loss of beads, break weak protein interactions... to use will vary between capture antibodies and can be determined through titration. Unbound or weakly... are removed through washing, the target protein can be eluted. The elution method will vary depending...

  7. Antibodies 101: Producing Recombinant Antibodies

    Type
    Blog Post
    ...rAbs. Production Recombinant antibody production can take place in bacterial, yeast, plant, or mammalian...faster-growing than mammalian cells, but mammalian cells can perform human-like post-translational modifications.... (L'Abbé et al., 2018).  Recombinant antibodies can be produced through a single or dual-plasmid transfection... The antibodies bind the immobilized ligands and can be eluted once the other materials have been washed...preferred buffer. If that’s the case, a buffer exchange can be performed using a desalting column or an ultrafiltration...extinction coefficient of IgG is around 1.35, though this can vary depending on the specific amino acid sequence...protein standard. The density of the antibody bands can be measured against the protein standard and the ...

  8. Tips for Writing a Good Cover Letter

    Type
    Blog Post
    ...relocation plans. They can be skills-based: if you're switching fields, you can use your cover letter ...cover letters can be a barrier for qualified applicants. In short, while a cover letter can’t replace skills...reproducible assay. That information can make you a much stronger candidate!  Additional uses The cover letter...transferable skills make you a good candidate for the position. You can also address any missing experience...cover letter won’t hurt your application, but it can certainly help you stand out.  In some fields, a ... application. Even within an organization, there can be variance. At Addgene, our content team, where ...have ddPCR experience. In your cover letter, you can expand on that by describing how you developed and...

  9. When is a Monomer not a Monomer? The Top Three Ways Your Favorite Fluorescent Protein Oligomerizes in Cells

    Type
    Blog Post
    ... that dimerize can form dynamic contacts that bring apposing membranes together and can wrap the membranes...noncovalent dimers. This property of EGFP can lead to significant artifacts. If you're using green fluorescent...families, which cannot form heterodimers (i.e. EGFP and mCherry). These simple strategies can help you make...homogenously distributed throughout the volume and can freely tumble in 720°. However, if the molecules ... FRET biosensors that contain two dimerizing FPs can exist in an open inactive state, a closed active ...FPs will collide with each other. The consequences can be dramatic. For a membrane POI localized to the ..., the normally spiderweb-like pattern of tubules can be grossly distorted into dense stacked membranes...

  10. Addgene’s Blog Gets a New Look!

    Type
    Blog Post
    ...present the vast collection of material so that you can more easily find what you’re looking for. Whether...Vectors Fluorescent Proteins Science Careers You can use the dropdowns in the menu to further drill down...cover topics outside of these five areas and you can find what you need by field (ex: microbiology, neuroscience...or publishing, under the Other dropdown menu. You can also find information on using Addgene’s website,...new to the topic. Or, try the search bar Now, you can search for the articles you want to find without ... them? Check out the new author pages, where you can read more about the Addgenie behind the blog post...

  11. What's Your Organism? Expanding Genomic Tools via the NSF EDGE Program

    Type
    Blog Post
    ...nests so can be studied over time. They recognize individual faces (I guess of other wasps) so can be used...This black footed slipper snail can be manipulated by injected RNA and can express fluorescent proteins....organism, but researchers are optimistic that this can be done for most, if not all, of these new experimental...is designed to expand the number of systems that can be studied by providing support "for development ... and educational programs for K-12 students. You can see why I was excited to be invited to attend. My...reagents from these emerging organisms so scientists can easily find the useful new reagents that come out... nest on your house. Their nests are open so you can see them and even feed them RNA to do silencing in...

  12. Antibodies 101: Reading a Flow Plot

    Type
    Blog Post
    ... variations in how one can gate, select, and analyze data. But now that you can read a FACS plot and follow...literature. Data reported from flow cytometry experiments can be a little challenging to understand if you’ve never...them it's helpful to have done flow first… which can send you in an endless loop. Let’s break out of the...users to analyze single cells in a population. It can be used to measure protein expression, identify rare... by the researcher, based on their controls. You can usually tell where this point is by looking at the...the cell populations as a guide  (Figure 3.) You can see that in our dot plot, we have A-B- cells, A-B...markers. There are various types of SSC and FSC that can be used for this purpose.  Pro tip! The forward scatter...

  13. CRISPR 101: Which Cas9 Do I Choose for My CRISPR Experiment?

    Type
    Blog Post
    ...overview of the various ways in which you can verify your genome edit can be found in our blog post entitled ...favorite gene. With so many Cas9 reagents available, it can be difficult to decipher which one is right for your...target for Cas9 and the introduction of this mutation can be any site early in the coding region of the gene... exons’ worth of DNA. Any ∼20 nucleotides of DNA can serve as the target for Cas9’s single guide RNA (.... Information on even more alternative PAM Cas9s can be found in “The PAM Requirement and Expanding CRISPR...maybe to disrupt a catalytic residue. Good news! You can achieve your precise edit without even making a break...protospacer). While this is a small window, this system can be used with a variety of impaired Cas9 proteins ...

  14. Getting the Most from Your Lentiviral Transduction

    Type
    Blog Post
    ...your target cells. Poor tissue culture practices can lead to low titers and poor transductions. Cell lines...serum, other cell lines, or infected personnel and can persist undetected; unlike infections with larger...microbes such as yeast, fungi, or bacteria, mycoplasma can be extremely hard to detect with levels reaching ...Mycoplasma compete with host cells for nutrients and can alter expression of receptors, ion channels, and ...and behavior. Signs of a mycoplasma contamination can be subtle and may include reduced saturation density... that extreme changes in osmotic pressure and pH can destabilize the viral envelope. One drawback of the... consider concentrating your viral prep. Storage can also play a role in the success of your lentiviral...

  15. Using Phosphoserine to Study Protein Phosphorylation

    Type
    Blog Post
    ...events in the proteomes of higher mammals (2). How can we begin to understand the importance of any of these...phosphorylation events on its activation loop, but can be made constitutively active through phosphomimetic...extensively worked with and optimized Sep-OTSλ and can recommend its use (6).  2. C321, a specialized bacterial... bacterial strain, TAG is an ‘open’ codon, which can be specifically recognized by the Sep-tRNA. 3. An...vector contains a mutant pSer-containing GFP that can be used as a positive control during optimization...PhosTag system (see below). This plasmid backbone can also be cloned with an ORF of interest for customized...non-phospho-incorporated proteins purified from the system, researchers can run samples on SDS-PAGE gels containing Phos-Tag,...

  16. 28 Hot Plasmid Technologies from 2015

    Type
    Blog Post
    ...functional Cas9 molecule can cleave the target DNA. The inducible split cas9 approach can also be used for activation...Finally, the system can be adapted to cellular screening to identify molecules that can perturb protein transport...through Addgene so you can try these incredible new tools yourself! The tags can be found in the pBluescript...that this tool can also be used to visualize translocations, genome rearrangements and cancer-associated ...shoot us an e-mail at [email protected] and maybe we can write a blog post about it! Split Cas9 system The...expression. The plasmids associated with this article can be obtained through Addgene: Rapamycin-inducible...genes are turned on or off in a given area and the can do so in a very specific, precise manner. Previously...

  17. Your Top Requested Plasmid in 2016!

    Type
    Blog Post
    ...particular backbone can be used to transfect a variety of mammalian cells and can be modified for viral...a versatile mammalian expression vector that you can use to express both SpCas9 and your gRNA in a variety...followed by BbsI restriction sites into which you can clone your 20 nucleotide guide sequence of choice...fused to a 3X FLAG tag at its N-terminus. This tag can be used to test for SpCas9 expression post transfection...a versatile mammalian expression vector that you can use to express both SpCas9 and your gRNA of interest... better facilitate homology directed repair. You can find all of these variants, as well as links to the...

  18. Developing Transferable Skills During Science Training

    Type
    Blog Post
    ...much as you can), form a peer mentoring group to get real feedback Write whenever you can, find supportive...Joanne's Reddit AMA Here are some concrete things you can do to develop those transferable skills while you...Demonstrating your ability to work well with others can be done in many ways… and many labs in academia benefit...advance science faster. Help other people when you can. The colleagues you help will be future advocates... team productive. Take all the opportunities you can to be communicating about science with others. If...organizations Another excellent perspective on this topic can be found on the UK Career Site for Researchers called...

  19. A Practical Guide to Optimizing AAV DIO and FLEx Vector Expression

    Type
    Blog Post
    ... and leaky expression can occur in cells that don’t express Cre or Flp, what can we do about it?  Test...comprised of different sequences (loxp and lox2272) and can not recombine with each other. When Cre is introduced...either end of the transgene. Since loxp and lox2272 can not recombine, this ensures that the transgene is...They found that almost all off-target expression can be attributed to spontaneous recombination between...This indicates that the amount of leaky expression can largely be attributed to the level of spontaneous...vectors will have some level of recombination that can lead to leaky expression in cells that do not express...genomes contain a recombined/functional transgene that can be expressed in cells where Cre is absent. Additionally...

  20. The Scientific Conference Poster Session: Tips for Success

    Type
    Blog Post
    ...and PI can provide feedback on your technical presentation, but those outside of your field can point ...Tailor your language to your audience so that they can digest what you’re saying, provide constructive criticism...determine how to tailor your presentation so that it can be understood by your audience. Use your poster as... aid, not a crutch. Even the most prepared of us can find ourselves talking to our posters, so if you ...aside to read through the list at the session so you can strategically use your time. Don’t have access to...yourself up for success. But sometimes, attendees can get sucked in to thirty-minute-long presentations...presentations, which constrains the number of posters you can see. If you are pressed for time, limit yourself to...
Showing: 301 - 320 of 1075 results