...Plasmids 101 posts here. Now that we have covered antibiotic selection here at Plasmids 101, we can talk about...amino acids (called the α-peptide) to a lacZΔM15-mutant bacterial cell in trans complements the mutation...they should be. Don't rush the process: It is important to give your plates enough time for any intact...identify colonies that not only have your plasmid (antibiotic resistance), but also confirm those plasmids ...α-peptide DNA disruption in the blue-white screen. Antibiotic selection is also used in conjunction with this...select for plasmid-containing cells without using antibiotic resistance. These rely on cell lines susceptible...

...considerations. First, it is important to establish the type of audience you want to reach with your paper... your paper to your preferred  journal, it is important to check the journal’s guidelines to authors and...catalogue numbers and manufacturers’ details for antibodies, plasmids (like your Addgene plasmid #), and ...an identifier (a bit like a GenBank ID) to all antibodies, animal models, and software tools in the Materials...journals and their scientific communities aim to significantly improve the reproducibility and reliability ...

... ask” once you have found someone you admire and want to learn from. Check out Joanne's Reddit AMA Who... colleagues that have the training or skills you want to learn. Mentors can be senior to you or peers ...mentors from your current organization but also important to build relationships outside your organization...confidential topics Broaden your network Take advantage of formal programs A formal mentoring program ...mentoring circle. This type of program has many advantages and I am seeing a lot of success at universities...feedback. The best thing to do to make your mentor want to keep working with you is report back with progress...

...transcription factors with important roles in Multiple Myeloma), it is important to ensure lack of degradation...Remarkably, small molecule degraders provide distinct advantages over pharmacological inhibitors including rapidly... are happy to share aliquots of dTAG-13 in the meantime, please address your requests for dTAG molecules... chimeras For each protein-of-interest, it is important to determine which terminus can accommodate the...context. Immunoblotting is recommended, while quantitative proteomics experiments will provide the best...

...Accelerate Research Discoveries) Challenge provides grants for rare disease research. This year is our fourth... and induce their effector functions. It’s an important process that we need to understand so that we ...source of IL-36 cytokines in the skin making it important to study what is happening within the keratinocytes...genes from the X chromosome are expressed at significantly reduced levels in women with TS (Trolle et al...nucleus,” says Dr. Terry Hébert, a professor and assistant dean of biomedical science education at McGill.... So far, there have been thirty GNB1 missense variants identified from 58 individuals with GNB1 disorder...

.... 1). When you have to tell an engineer that you want the LIMS software to create a barcode for your plate...each given container, when applicable Any other relevant information Prescribing an official name to all... all of our plates, tubes, and racks meant we had fewer errors in our LIMS tracking system. Though you...terminology of a new field. Annotate: When creating important documentation, including SOPs or project-related...Product Manager role at Addgene is “Words are important!”  If you try any of these approaches in your ...propensity for cross team communication, that’s an important soft skill to carry into the next phase of your...

...on the conversion of non-malignant forms of cancer to invasive and malignant forms and noticed that myoepithelial...stromal cell modifications, stromal cells are also constantly communicating with a tumor, proving once again...malignancy but also incite different organs to become malignant. Exosomes also contain miRNA (micro RNA) and other... case, exosomes may transport miRNA from drug-resistant cells to sensitive ones. Though this process is..., the microenvironment backstage play just as important of a role if not more.  Many thanks to our guest...

...the brain. “We hope that iGluSnFR3 will enable significant steps forward in our understanding of synaptic...Filip Tomaska and Tim Hanson, are characterizing variants of iGluSnFR3 in vivo and have used it to detect...hydropersulfides modulate protein activity and have important roles during oxidative stress. To monitor this...properties. Her method takes a pooled library of mutant sensors expressed in a mammalian cell line engineered...engineered to be electrically excitable. The mutants are co-expressed with a photoconvertible fluorescent...photoconverted, sorted, and sequenced to identify the mutants for further enrichment. Tian notes that this platform...

... and Timothy K. Lu. "Enhanced killing of antibiotic-resistant bacteria enabled by massively parallel combinatorial...particular phenotype, and quickly profile the resultant hits (Cheng et al., 2014; Wong et al., 2015; Wong...plasmid. Each gRNA combination can be tracked and quantitatively analyzed by sequencing its set of barcodes....efforts made by various research teams to build and constantly improve gRNA libraries, excellent resources with... Wong et al 2015. Alan Wong is currently an Assistant Professor at the School of Biomedical Sciences...

... nuclease-resistant, resulting in less off-target events. While both have similar advantages, bridged ...Nature has evolved ways to prevent degradation of important cellular RNA molecules with post translational...-Cas9, for use in applications like siRNA and antisense oligonucleotides. Some of these synthetic modifications...Frontiers in Genome Editing). This is particularly important in experiments in which viability is being compromised...technology is that most expression systems are constantly “on”. There is no mechanism to turn cutting off...

...high-intensity light for activation which requires implantation of fiber-optic cables into the brain. ...information about parental channelrhodopsins to create recombinant channelrhodopsin sequences. The model first removed...performed well in a tissue culture dish, that doesn’t guarantee their success in vivo. Therefore, the Arnold and...fiber-optic cannulas were still stereotaxically implanted into the mice’s brains so that light could reach...into a hole in their cage. Since dopamine is an important neurotransmitter that promotes reward-motivated... brain may still require fiber-optic cannula implantation, but altering ChRgers so they can be activated...

...number of important plasmid elements – promoters, origins of replication, protein tags, and antibiotic resistance...recombination at the loxP recognition sites. A key advantage of the system is that Cre acts independently of...potentially recombine. To account for this, alternate mutant versions of the loxP site have been created, which...lox2272 (GgATACtT), and lox511 (GtATACAT). These variant lox sites undergo recombination with other sites...not cross-compatible. Using different lox site variants allows Cre to catalyze more than one specific ...

... of the fusion protein. Advantages of Split-BioID Split-BioID’s key advantage over other PPI methods, ... lysate fractionation. Important considerations for Split-BioID It’s important to remember that with Split-BioID...proximity-dependent biotinylation technique BioID. The key advantage of Split-BioID is that it allows for the validation...bait protein is pulled down, typically with an antibody. This can artificially select against PPIs that...

...expression, plant expression, mouse targeting, CRISPR, TALEN, synthetic biology, affinity reagent/antibody, unspecified...be indicated in the “Species Other” column. Relevant Mutations Indicate the amino acid change as ...other. Bacterial Resistance Enter bacterial antibiotic resistance encoded in the plasmid that should...or low copy. Choose “high copy” if a sufficient quantity of DNA is produced from a miniprep and “low copy...Pro Tip! Download a new spreadsheet each time you want to submit new plasmids! We update the spreadsheet...

... series). In any case, Cre is a dominant phenotype, while you’ll want the floxed gene to be homozygous...these strains should be crossed to generate double-mutant offspring. Crossing mice allows for the production...homozygous for one of the two mutations that you want combined in your offspring. Genotyping involves ...generations. 4. Continue breeding your colony You’ll want to generate enough mice that are homozygous (or ...require several rounds of breeding in which you’ll want to genotype your mice for expression of Cre, loxP...

...expressing variants of Yellow Fluorescent Protein (YFP). The team identified a new YFP variant, mGold. mGold... were exposed to blue light. Cre-Vvd showed substantially improved activity as compared to other recombinase...CRISPR-Casɸ is active in vitro as well as in human and plant cells. Casɸ from Biggiephage can eliminate...Birsoy. Find the library here A new SaCas9 ABE variant (microABEI744) has improved on-target editing efficiency...compared to current N-terminal linked SaCas9 ABE variants. It's also one of the smallest AAV-deliverable...

...updated our blog post on Prime Editing, and that meant rereading many of the original papers reporting ...editing application. And optimizing its sequence is important, as prime editing efficiency is affected by the...nucleotides. For longer templates, it is even more important to test different lengths and sequences, as unintended...whose 3′ extension begins with a C base can cause unwanted base pairing (left), while pegRNAs that use other... Tool (pegLIT) to create linkers that minimize unwanted intra-RNA base pairing with the primer binding...prime editing systems by manipulating cellular determinants of editing outcomes. Cell, 184(22), 5635-5652...

...weeks designing and cloning the vectors you actually want. But now you can use the recently developed third...website, users can simply select the components they want for each module, generate the vector file, and quickly... license.   Once you have the components you want, simply assemble them into a destination vector ...vectors are available? What you’re building is important — but so is where you’re building it! Table 1 ...selections) Module set (the set of components you want in your vectors) Target type (drop down options...kit. What if Fragmid doesn’t have the components I want? You can make your own Fragmid components! Ensure...

...when the hood is in use. Wipe the hood with a disinfectant (most people use 70% ethanol, but you can also...wipes) before and after your work. Keep your contaminants away While it’s likely that you’re the main ...pipettes into solution bottles directly, rather decant the solutions into disposable sterile tubes first...already be at 37 °C. Most labs have a water bath constantly at 37°C for this purpose.Learn about the effects...microscope will help you identify bacterial and yeast contaminants. Set up a routine to split cells Don’t be lazy...passage are too young and should not be used for important transfection experiments right after they’ve been...

...chromoproteins and fluorescent proteins, it’s important to note that despite their distinction by name...photosynthetically active wavelengths to reach zooxanthellae, the coral’s symbiotic dinoflagellates that ...be used for mimicry. Image: Bernard Spragg. Advantages of using chromoproteins for molecular biology...bacteria, where most genetic manipulations start. Anthony Forster’s lab used codon optimization programs ...interactions with cytosolic proteins. This step is important as non-specific binding to proteins can inhibit...Characterization of a Blue Chromoprotein and Its Yellow Mutant from the Sea AnemoneCnidopus Japonicus. Journal...