...have stable ssDNA secondary structure — for example, the stem-loop structure of transcriptional terminators...higher temperature at which Gibson assembly takes place may limit formation of secondary structures at the...with 5′ overhangs generated either by annealing a mixture of products or from incomplete PCR — although higher...effective with as little as 3 ng of DNA!) Figure 1: Schematic of SLIC. To start the SLIC cloning...successful, but at a lower efficiency (~20%). Figure 2: Schematic of multicomponent assembly with SLIC...

...surface of most immature B cells, its function is unknown. It has a monomer structure with a valency of... region: IgM, IgD, IgG, IgE, and IgA.    Figure 1: Diagram of an antibody labeled with Fc, Fab,...antigen of interest. Isotype controls are used to make sure the staining you see is due to target-specific antibody...region length. Now for some essential isotype nomenclature. Antibody heavy chain proteins as well as the...cells begin by expressing IgM. IgM has a pentamer structure, which means each IgM molecule has a binding capacity... for IgM, IgD, and IgG are deleted.     Figure 2: Isotype switching requires DNA recombination...

...synthetic) pFZ19 (yellow flame) Temperature inducible promoters Temperature sensitive expression systems ... expression at regular temperatures but can be induced by heat or cold exposure. Examples include the ...Types of inducible promoters Chemical agents, temperature, and light are all examples of factors that can...which a gene of choice is only expressed following exposure to a brief heat shock. In the case of Hsp70, the...since expression can be repressed by glucose. Temperature inducible promoters are also known for their ...

...sandwiched between 2 loxp sites and is excised by Cre (Figure 1). This results in one loxp site and one lox2272...Since loxp and lox2272 can not recombine, this ensures that the transgene is no longer able to flip back...the cause of this spurious expression?   Figure 1.  Cre-recombinase binds to either pair of lox...orientation preventing the transgene from reverting. Figure created with BioRender.com   Causes of recombination...to flank the transgenes (non-homologous pairs) (Figure 2.). They found that almost all off-target expression... to exercise caution when using them.    Figure 2. Schematic diagram showing the mechanism of spontaneous...reduced reversion and less off-target expression. Figure created with BioRender.com   Strategies for...

...University of Manitoba. Location, Location, Location! Failure to Plan (for Storage) Is Planning to Fail Patience...Patience Is Bitter but Its Fruit Is Sweet The Future? I knew little about adeno-associated Viral Vectors...sufficient to overcome the transport issues. 2. Failure to plan (for storage) Is planning to fail Freeze...not take much time at all. Much of the previous literature using AAVs for DREADD delivery indicates ~7-21...to use AAV constructs with self-complementing structure (scAAV) (7). As single stranded DNA viruses, AAVs...from ~4.8 kB [8] to ~2.3 kB [9]).     4. The Future What new types of research may be enabled by the...experiments, a group of researchers has identified this feature of AAVs as a new tool for neuroanatomical tracing...

... a native peptide bond at the split site. Figure 1: The base editor is split into two plasmids and...the split base editors at Addgene. Levy et al., Nature Biomedical Engineering, 2020. PubMed PMID: 31937940...in higher quenching of mScarlet emission. Figure 2: Ace-mScarlet is a fusion between the voltage-sensitive...synthetic biology tools for cyanobacteria. Figure 3: Assembly of plasmids using the Cyanogate Kit...reducing Cre toxicity in long-term experiments.  Figure 4: The C-terminus of Cre (CreC) and the N-terminus...(PCB), is required for PhyBΔ to be functional. Exposure to red light induces PhyBΔ and PIF6APB binding...This novel tool is practical in cell and organ culture, as well as cell lineage studies. Eisenhoffer and...

...engineering enzymes, each with their own unique features and quirks. Here, we will review the genome engineering.... Dozens of variants exist with unique cutting features and evolved PAM sites, making Cas9 an adaptable...PAM does not require any G’s or C’s. One curious feature of this Cas is that once it is engaged by its target...target, it will non-specifically cut ssDNA. This feature is shared by other Cas enzymes outlined below as... proteins should not be overlooked. The unique features and targeting abilities of the other Cas family...Janice, et. al. Programmed DNA destruction by miniature CRISPR-Cas14 enzymes. Science, 362(6416): 839-...with the single-protein CRISPR effector Cas7-11. Nature, 597, 720-725 (2021). 10.1038/s41586-021-03886-...

...Glutamate sensing reporters Abhi Aggarwal is a postbaccalaureate research technician in Kaspar Podgorski’s ...expression and trafficking of these biosensors.   Figure 1: Schematic of iGluSnFR3 (left). GluSnFR3 in pMinDisplay... domain), in electroporated in rat hippocampal cultured neurons, imaged 14 days post-transfection, excited...happen inside the cell,” says Pedre Pérez. Figure 2: Micrographs of the biosensor expressed in the...from opioid use disorder,” says Hubbard. Figure 3: 20x confocal image of the periaqueductal gray...response to light touch in mechanical allodynia. Figure 4: While Qiu’s work on the PV neurons is in progress...calcium, and membrane voltage,” says Tian. Figure 5: Citrine-tagged voltage indicator expressed in...

...the analysis did find 263,000 more osteoporotic fractures. The overall economic savings to the US healthcare... Unfortunately, it also meant that, by the very nature of the way the experiments were performed, they...and cell research. Using cells from both sexes ensures that the diagnostics and therapeutic interventions...recent years, and you can see a summary of this literature in our health and medicine checklist. A common...not analyzed) may harm humans or contribute to failures in drug development – costs far greater than those...doing the research correctly from the get go. The future of sex and gender in research There is much work...Blog Learn How Scientists Are Trying to Shape the Future of Research Learn How Notions of Scientific Impact...

...we’re really borrowing these tools from Mother Nature’s toolbox. Natural plasmids, also called environmental...the basis of many molecular biology tools! Figure 1: Common functions of natural plasmids. Created... to survive where others can’t. This ability to endure a harsh environment is distinct from the benefit...new types of plasmids without clear analogues in nature. For example, helper plasmids were developed to... To learn more about some other common plasmid features, such as origins of replication and promoters,... blog as scientists continue to explore Mother Nature’s toolbox for new and exciting molecular technologies...Resources References Demanèche, S., Bertolla, F., Buret, F., Nalin, R., Sailland, A., Auriol, P., Vogel,...

...Did you say… easier? Fragmid’s modular assembly structure takes a lot of the design work out of the plasmid...Fragmid components on the Fragmid website.   Figure 1: (Top) Schematic overview and timeline of the...schematized. Sizes of fragments are not to scale. Figure and caption from McGee et al., 2024, used under...designing a CRISPR vector the Fragmid way!    Figure 1: Selecting your vector type on the Fragmid website...website.   In step one, you’ll select the features of your vector, which are: Organism (Mammalian...mechanism (KO, CRISPRa, CRISPRi, editing)   Figure 2: Selecting module fragments on the Fragmid website...want? You can make your own Fragmid components! Ensure your backbone and genes contain only the Bbsl cut...

...(ITR) present at both ends.   Figure 1: A) The structure of an adenovirus: the virion is ~ 100nm...genomes/mL) can be measured by: Optical density: particle concentration is measured by looking at absorbance... construct from step #1 and allowed to stay in culture for up to 20 additional days at which time the ...recombinant adenoviruses using the AdEasy system." Nature protocols 2.5 (2007): 1236. PubMed PMID: 17546019...production of high-capacity adenoviral vectors." Nature protocols 4.4 (2009): 547. PubMed PMID: 19373227...

...Met protein complementation. In the procedure diagrammed in Figure 3, students first transform the S. ...available (6). Methionine synthesis in S. cerevisiae (Figure 1) occurs through a well-characterized pathway ... the deletion). Find the Plasmids Here Plasmid features enable multiple student experiments We used TOPO...coding sequences for ten different MET/Met genes (Figure 1) from the two yeast species into the pYES2.1/... controlled by the GAL1 promoter. The cloning procedure adds both V5 and His6 epitope tags to the C-termini...expression by western blotting in other experiments (Figure 2).   Complementation reveals conservation of function...

...office or other authorized office directly to make sure the deposit agreement/MTA is taken care of and, ...Deposit” and “Download Deposit Spreadsheet” (see Figure 1). Please read our “How to Deposit Your Plasmids...thorough guide to our online data entry process. Figure 1: Begin the deposit process by clicking “Submit...Required columns are shown in red in the spreadsheet (Figure 2) and are described in Table 1. Some cells have...Growth Temp Choose from 30 °C, 37 °C, or room temperature. Growth Strain Please indicate which growth...Filling out and sending the deposit spreadsheet Figure 2: Example of the deposit spreadsheet showing the...update the spreadsheet every so often and this ensures you are using our most up-to-date version! We hope...

...focus was on editing animal models of disease and cultured cells. For example, in 2013, Jinsong Li and colleagues...When all was said and done, the researchers had cured 24 mice of their disease. Meanwhile, Hans Clevers...significant progress in clinical trials.     Figure 1: Types of delivery and editing strategies for...success rate in clearing misfolded protein levels as measured in the blood. More information is expected soon...blindness, and diabetes. Directions and obstacles for future CRISPR therapies CRISPR technology is rapidly evolving...strides since we first wrote this blog post. The future success of CRISPR in the clinical will rely on ...MA, Berkhout B, Herrera-Carrillo E. A CRISPR-Cas Cure for HIV/AIDS. Int J Mol Sci. 2023 Jan 13;24(2):1563...

...individual parts are reusable, saving time and money on future assemblies. If you are new to MoClo, give our MoClo...synthesis of these molecules. To make large amounts of pure product in an affordable way, researchers test many...your yeast experimental goals are more general in nature, or you aren’t planning on using K. phaffii, then...proteins within the mitochondrial matrix.     Figure 1: A chart with MoClo kit recommendations for different...2023). The Plant Parts II kit has a focus on infrastructure with shuttle vectors, several delivery systems...targets or multiple sgRNAs against the same target to ensure efficient cutting. Beyond sgRNAs, MoClo can also...convolute the results of these experiments. The modular nature of the Fragmid system allows for direct comparisons...

...Behavioral experimentation I’ll discuss some of the key features of each of these steps. Today we’ll discuss selection...relies on identification of the bregma and lambda sutures and subsequent determination of the proper coordinates... your brain region of interest (as shown in the figure below). Since most of the precision of optogenetics...circuits underlying brain disease in animal models. Nature reviews Neuroscience. 2012;13(4):251-66. doi: 10.1038...of neuroscience. 2011;34:389-412. doi: 10.1146/annurev-neuro-061010-113817. PMID: 21692661.  3. Bernstein...direct comparative analysis of microbial opsins. Nature methods. 2012;9(2):159-72. doi: 10.1038/nmeth.1808...mice for light-induced activation and silencing. Nature neuroscience. 2012;15(5):793-802. doi: 10.1038/...

...electrical potential. Thankfully, there is a tool to measure these electrical events! Voltage indicators are...They can be targeted to the plasma membrane to measure membrane potential and can also be directed to ...applications such as in vivo tissue imaging.   Figure 1: Ace-mScarlet is a fusion between the voltage-sensitive...depending on your experimental system.   Figure 2: General overview of chemigenetic tools. A genetically...vitro and a small molecule is added exogenously to measure a phenotypic response in the form of fluorescence...Indicators As previously mentioned, cellular voltage is measured at the cell membrane, and this voltage is particularly...has you covered. Happy voltage sensing!   Figure 3: Components of the types of voltage indicators...

...the CRISPR/Cas9 system. Nature 516:423–427 . https://doi.org/10.1038/nature13902 Maggio I, Holkers M, Liu... the target cells (Beatty et al., 2012). These features make AdV an attractive candidate for in vivo delivery...genome editing machinery such as CRISPR-Cas. Figure 1: Schematic of targeted gene knock-out (left) ...Gonçalves' lab found in a second paper published in Nature Methods the same year that delivering RNA-guided...double-stranded AdV genomes. Such protein-DNA structures presumably reduce the likelihood that donor DNA... that advantages of AdVs include their episomal nature and very efficient introduction of DNA into therapeutically...

...compatibility and high DNA specificity." Nature (2018) doi: 10.1038/nature26155 PubMed PMID: 29512652 Plasmids...loci or genomes. Cas9s from other species, like S. aureus and N. meningitidis require different PAM sites...first restored catalytic residues D10 and H840. To ensure that the clones were capable of cleavage, they ...fused to third generation cytidine base editor architecture (BE3) slightly improves NGG PAM editing efficiency...efficiencies (edit frequency in cell population measured with NGS) are shown as percentages, with fold ...lot we still don’t know about xCas9. “I’m not 100% sure xCas9 is going to be flat out better than SpCas9...