...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...

... 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...

...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 ...

...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...

... 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...

...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...

...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...

... 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...

...or the recombinant antibody can be purified from the supernatant (see our Recombinant Antibody Purification...for Recombinant Antibodies You may also like... Antibody Plasmid Collection Antibodies Blog Posts Molecular...inhibitor cocktail to the supernatant. The supernatant containing the recombinant antibody can be used as-is ...cells to produce recombinant antibody. Protocols... Protocols Transfection for Recombinant Antibodies Transfection for... Reference General Transfection Protocol Recombinant Antibody Purification Protocol Introduction Transfections...to transfect suspension HEK293 cells with recombinant antibody plasmids using Polyethylenimine Max as a...

...identical molecules of DNA. Gene targeting takes advantage of this natural process to replace a targeted ...marker HSV-tk is used to select against random recombinants.   Design your targeting construct. For...been cloned into the vector on either side of an antibiotic resistance gene. To avoid selecting for cells...will first perform a positive selection for the antibiotic resistance gene and then a counter selection ...Remove the positive selection marker. Since an antibiotic resistance gene can affect a cell's phenotype...a true knockout can be lethal, and you'd instead want to create a conditional knockout. To make a conditional...

... expressed proteins. Here we will review what advantages degron tags can offer over other protein control... protein expression. These tags offer several advantages over the alternative systems – CRISPR, siRNA,...function, the SMASh tag self-cleaves itself when you want the protein around, leaving a minimal additional...with the most alternative functions – there are antibodies to it and ways to achieve fluorescence with it...might be the way to go (dTAG/SMASh). If you don’t want to deal with additional components beyond the minimum...Plasmids for Endogenous Gene Tagging in Human Cells Antibodies 101: Epitope Tags  ...

... cells. To study this phenomenon you will likely want to monitor the voltage change across the membrane...comparable to its one component counterpart and is significantly brighter and available with red-shifted fluorophores...for penetrating deeper tissues depths (1 mm), important for certain applications such as in vivo tissue...encoded component of the system to offer unique advantages depending on your experimental system.   ...and this voltage is particularly physiologically relevant in neurons, myocytes, and endocrine cells. It ...sensor selectively is especially essential. If you want to learn more about how viruses can be used for ...

...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...

...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...

... between the microhomologies.MMEJ has certain advantages over HDR. Some species don’t have good HDR systems...locus-specific gRNAs Step 4: Select for puromycin resistant cells Step 5: PCR amplify and sequence the locus...into the FBL locus. Upon sequencing of puromycin resistant clones, they found that 80% and 50% of clones ...technique can be adapted to other transgenes. It’s important to note that the fluorescence level observed will...follows. For increased versatility, it would be advantageous to adapt PITCh to insert genes into AAVS1, the...to insert cDNA constructs into this locus. One important question to ask is how the efficiency of PITCh-based...

... to achieve: Do you want to permanently knock out or knock-in a gene? Do you want to activate or repress...trying to create a single point mutation? Do you want to create a fusion with a reporter protein such ...sufficient? Which cell types will you be editing? Do you want to deliver the components as DNA, or would mRNA ...and Cas9 are required for any application. If you want to include a homology directed repair (HDR) template...well characterized in terms of CRISPR use, you will want to invest some time in optimizing and testing the...

...vector that encodes both Cas9 and sgRNA. What if I want to work with gene drives? Then do so! It's a tremendously...that anyone else who came up with the same idea or wanted to build a gene drive in a model organism would...experiments2,3. While that didn't work quite as well as anticipated4, greater awareness will certainly improve safety...regions that don't have resident populations of the relevant mosquito species. Reproductive confinement involves... cas9 gene). An immunizing reversal drive is a variant that also spreads through the wild population and...gene drives. Science 345, 626–628 (2014). PubMed. Gantz, V. M. & Bier, E. The mutagenic chain reaction: ...

...Addgene's Antibody Guide Addgene's Ready-to-Use Antibody collection Addgene's Recombinant Antibody Plasmids...sure that the sample is pure and free of any contaminants that could affect the measurements. You'll need...below 0.95! More resources on the Addgene blog Antibodies 101: ELISAs The Four ELISAs and When to Use Them...

...the cells’ germline genetic variations. They then wanted to identify cancer-associated genetic dependencies...ancestry, Sean and Aaron knew that their study had significant statistical limitations, and expected to find...Cas9 from cutting, both of which can introduce significant bias into the results. But the reference genome...design of the experiment or dataset itself. It is important for scientists to not only be aware of the potential...ancestral bias and the human genome Wang, T., Antonacci-Fulton, L., Howe, K. et al. The Human Pangenome...doi.org/10.1038/s41586-022-04601-8 Lewis, Anna CF, Santiago J. Molina, Paul S. Appelbaum, Bege Dauda, Anna...