...before their paper can be published. To a scientist, all of this checking and formatting can feel like yet ...lot of time. It doesn’t have to be this way. We can never completely eliminate the risk that a minor ...stability in an incubator), but there’s a lot you can do to help other scientists both what you did and...scientific citizen” and making sure you do what you can to help your fellow researchers advance science overall... Communication is the issue Much of the problem can be attributed to communication, and in particular...Admittedly, the differences are often very subtle, and it can take a lot of detective work to figure them out. ...accessible and consistent way so other researchers can use them, to making sure that methods and protocols...

...Viruses are the SUVs of molecular biology – they can deliver materials to specific destinations, haul ...research all the way to clinical gene therapy. But how can you use these amazing tools in your experiments? ...cargo is typically their own genome, but this step can be engineered to be anything you want delivered to... to your target cells. This delivered DNA/RNA can then be either permanently integrated into the genome...application, our Viruses as Biological Tools blog can help you learn about the different types of viruses...degradation. Viral introduction of shRNA plasmids can induce gene-specific silencing temporarily, long-...turning expression off permanently. Viral vectors can also be used to express proteins. Viral expression...

... way, you can run one PCR, one gel, etc. for multiple plasmids at once. Over time, this can save you a...how much time you have to spend cloning before you can start your experiment? What about all the reagents...eternity of cloning, waiting, and repeating before you can finally dive deep into generating the data you’re...Table 1). Restriction enzymes and vector purchases can raise the cost anywhere from a few dollars to over...varies hugely based on a number of factors, but we can assume it’s at least equal to the $155 spent on reagents...short on money or time, there are a few options you can explore to maximize the use of both when making plasmids...) Ask the PI If you want a plasmid for free, you can always reach out to the PI or corresponding author...

... 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...

...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...

...Preprints can be read freely by anyone and are typically free of charge for authors to post. They can be assessed...materials available to the community significantly sooner. Plus, authors can include the Addgene ID for their...them as best you can! At the time of writing, Mike Lacy was also employed by the American Society for Cell...sharing results faster and more broadly, scientists can receive recognition for their efforts, inspire future... preprint as evidence of their productivity that can be shared with hiring and review committees, as opposed...comments, reviewers’ comments, and author responses can be shared directly alongside the preprint while the...awaiting a final publication in a journal.  Preprints can be one tool in a combination of strategies for your...

...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...

...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...

...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 ...

...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...

...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...

...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...

... 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...

...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...

... 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...

...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 ...

...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...

...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...

...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,...

...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...