... it matter which antibiotic resistance gene you select? In this blog, we’ll break down the mechanism of...satellite colonies from forming while effectively selecting plasmid-containing bacteria. So… why do some people...which is commonly used for eukaryotic antibiotic selection. Essentially, this gene is a 2-in-1 providing ...providing both bacterial and eukaryotic selection, whereas other plasmids must carry both a bacterial and a mammalian...mammalian antibiotic selection gene. However, it does require a slower transformation recovery period ...bacteria used in research, it isn’t a widely popular selection agent. Pros: Stable (spectinomycin). Cons: Cost...is handy because it can be a one-stop shop for selection across systems and species. The cost? Zeocin is...

... start selecting and testing systemic capsids! Figure 2 contains a decision tree for selecting a capsid...C57BL/6J, the strain in which the capsid was first selected. AAV-PHP.B, and many capsids derived from it, ...particularly between New World and Old World monkeys. Selecting your systemic capsid Once you’ve decided that ...descriptions of commonly used or emerging capsids. Selected capsids are further highlighted below the table... the CNS. It was engineered, via the M-CREATE selection method, to cross the BBB through interactions ...the DELIVER (Tabebordbar et al., 2021) method, selecting for variants that were effective in C57BL/6J mice...et al., 2023). It was rationally engineered and selected for its ability to bind to the LY6C1 protein in...

...weeks (in vitro) or months (in vivo).  Selective promoters Selective promoters, such as synapsin, glial fibrillary...that can affect expression, before starting the selection process. Factors to think about are (1) your experimental....   Once you know that, you’re ready to start selecting, testing, and optimizing three key variables of... Figure 1: Flow chart depicting a) steps for selecting serotypes and promoters to test in your AAV system...be expressed; it can be ubiquitous, cell-type selective, or inducible.  Ubiquitous promoters  These often-strong...   Figure 3: Comparison of two astrocyte selective promoters demonstrating off-target expression ...validating your viral vector. Titer Once you have selected potential serotypes and promoters to begin testing...

...Gray labs, we synthesize compounds that enable selective removal of proteins-of-interest from the proteome...developed small molecule degraders that achieve selective degradation of endogenous proteins (notably, BRD2...depleting a protein-of-interest, increasing target selectivity, overcoming resistance to inhibitors, and inducing...apply this technique to oncoproteins that lack selective small molecule binders. To overcome this limitation...can use it to evaluate the effects of rapid and selective degradation of protein targets in time scales ... The dTAG system The dTAG system pairs highly selective small molecule FKBP12F36V degraders (dTAG molecule...performance, great depth of degradation, absolute selectivity, broad utility for degrading targets in different...

...Figure 1: Selecting your vector type on the Fragmid website.   In step one, you’ll select the features...components. Using the handy website, users can simply select the components they want for each module, generate...cassettes, promoters, N’ and/or C’ terminus, and selection markers. Need fewer components? A spacer can be...transgenic flies; gypsy insulator sequences; white + selectable marker; attB site for genome integration by phiC31...Destination vector (dropdown options depending on above selections) Module set (the set of components you want ...Target type (drop down options depending on above selections.) CRISPR enzyme (Cas9 [sp], Cas9 [other], Cas12a...CRISPRa, CRISPRi, editing)   Figure 2: Selecting module fragments on the Fragmid website. ...

... 2020). This selection platform was designed so that any standard lab can quickly select for sybodies ...steps to select for optimal sybody candidates. Figure 2: Outline of the process for selection of sybodies...our secondary nanobodies blog post.  A rapid selection platform for generating sybodies against membrane...a robust in-vitro protocol that enables rapid selection of synthetic nanobodies, called sybodies, against...The Seeger lab starts with a “pre-enrichment” selection step using ribosome display because it has the...using phage and yeast (109-1010 members). This selection step involves using a commercial kit for in-vitro...membrane-bound proteins. The full protocol for this sybody selection platform can be found here and relevant plasmids...

...bacteria due to site selection by TnsD, a sequence-specific DNA binding protein. Selection removes donor bacteria...colonies that grew on a selective plate versus the number that grew on a non-selective plate. Most bacteria...strain-specific promoters, antibiotic resistance selection markers, gRNAs, and dCas9 proteins. Second, it...recipient's genome. Antibiotic plates lacking DAP select against donor bacteria and for recipients with ...integrates into the trnS-leu2 locus. Antibiotics select for recipient bacteria with an integrated copy ...allows for the customization of guides, promoters, selection markers, and different versions of dCas9. Following...integration, Mobile-CRISPRi is stably expressed with no selection required and titratable knockdown allows for the...

...contain both a protein modification sequence and a selectable gene product for isolation of successful transformants...fluorescent tags, epitope tags, protease sites, and selection markers. These are especially useful in protein...multiple protein products is desired, as multiple selection markers can ensure that all desired tags have ... vectors and include a Kan, SpHIS5, or CaURA3 selection marker. Lee et al.(1) assessed many of these fluorescent...filter sets and experimental output requirements. Select from these yeast-optimized fluorophore tagging ...12xHis, 2xStrep, 3xFlag, Protein A, or V5), and selection marker (KanMX, HphMX, or His3MX).  Each PCR product...)-6xGly linker-(epitope tag)-ADH1 terminator-(selection marker). Additionally, Dr. Formosa has deposited...

...positive and negative selection in plasmid cloning.  But oftentimes, a selection strategy is not enough...don’t. One way to begin the search is by using selection strategies, where only cells that have gained ... killing off a portion of the cells like in a selection. Instead, all cells survive and you will need ...through them to find the desired clone. Do your selection, then screen Why add a screen to your cloning ...after your experiments. As a common example, a selection will leave you with the colonies that contain ...ampicillin resistance marker in the backbone. A selection on agar containing ampicillin will yield bacteria...numbers game. By using a variety of screening and selection techniques, you can increase the chances of finding...

... negative selection: labeling markers that your cells of interest do not have, and selecting for the cells...antibodies may exist, but only with a limited selection of fluorophores. If the available colors are already...great flexibility when it comes to fluorophore selection for panel design. Find streptavidin plasmids and...intensity. However, you need to be careful when selecting the primary antibody host species, as it has to...labeled. An easy and quick method of negatively selecting your cells of interest is magnetic bead-activated...your cells of interest via FACS using negative selection is called dump gating. Let’s say we want to sort... result, all cells positively labeled with the selected color will not be CD8+ T cells and we can simply...

...involved in neurodegenerative diseases, the Cole Lab selected promoters for cell types directly linked to disease...PLoS ONE. 2017. PubMed PMID: 28241009 CRISPR coselection with ouabain increases editing efficiency Blue...efficiency in mammalian cells. Their marker-free coselection, analogous to methods previously used in C. elegans... interest has also been edited! Thus, ouabain selection enriches the population for CRISPR edits and improves...cellular growth delays. With eSpCas9(1.1) and coselection, Agudelo et al. achieved editing rates as high...high as 83% with NHEJ and 40-50% with HDR. Coselection efficiency is so high that some applications may...efficiencies with AsCpf1, indicating that ouabain coselection is a robust and flexible way to increase CRISPR...

...at a fundamental level. Optimal Cas selection Once you’ve selected a gRNA with low sequence similarity...least one will likely be useful. Optimal gRNA selection With some CRISPR experiments, there is hardly ... wiggle room, you can minimize off targets by selecting a gRNA with low sequence similarity elsewhere ...of bases. To reduce off-targets, you’ll want to select a Cas enzyme with low tolerance for mismatches ...the gRNA can be sequenced. These sites can be selectively sequenced to determine if there are significant...experiment may not have flexibility with gRNA selection, or maybe you just can’t get them below a given...

...protein obtained. Selectable Marker The antibiotic resistance gene allows for selection in bacteria. However...lab-created plasmids have an origin of replication, selection marker, and cloning site. The ease of modifying...proteins Antibiotic Resistance Gene Allows for selection of plasmid-containing bacteria. Multiple Cloning...However, many plasmids also have selectable markers for use in other cell types. Primer Binding Site ...gene is transformed into bacterial cells and selectively grown on antibiotic plates.  Importantly, because...

... bacterial selection cassette for growth and cloning in bacteria and a mammalian selection cassette to...host organism, usually achieved with the proper selection marker or antibiotic resistance. Next, consider...transfected cells, you will not need a mammalian selection cassette (e.g. 1435 pSG5L Flag HA). You can also...yeast with an integrating vector expressing a selection cassette with 5′ and 3′ flanking sequences matching... several fusion proteins. Pick your promoter, selection cassette and visualization vehicle to conduct ...

...transfected. Alternatively, you can use a selection marker to select transfected cells (for example, plasmid...your HR template has a selection marker such as puromycin, you can (also) select for the positive colonies...the genome, how can cells with that mutation be selected/screened? A4: Before starting your experiment,...colonies by puromycin selection. You could then confirm this purification by performing a genotyping assay...

... entire process of antibody selection and maturation in vitro by selecting for antigen binding from libraries...libraries of recombinant antibodies. The selection process is often performed via phage display, yeast...mammalian display (Tsuruta et al., 2017). In vitro selection allows scientists to create antibodies to targets...Wright GJ (2010) A rapid and scalable method for selecting recombinant mouse monoclonal antibodies. BMC Biology..., Moro AM (2018) Display Technologies for the Selection of Monoclonal Antibodies for Clinical Use. In:...

...pooled library, blog posts, protocols, and more. Selecting crispr in Guides will return links including our... through 300+ plasmids. Under ‘Vector Types’ I selected ‘Lentiviral’ which eliminates most of the plasmids...show up! Plasmid filter categories Expression: Select the model system for your research (e.g. Mammalian...inserts Resistance Marker: This indicates the selectable marker used in your model system, and is different...choose the antibiotic resistance gene(s) used for selection in bacteria. Has Viral Service?: Allows you to...scientists Viral prep filter categories Promoters: Select the best promoter for your target cell type Serotype...

...some positive selection (ex: antibiotic selection and auxotrophy) and negative selection (ex: toxin-antitoxin...scientists. 9. Plasmids 101: Positive and Negative Selection for Plasmid Cloning by Jennifer Tsang There are...toxin-antitoxin systems and sacB counter-selection) tools used in cloning. 8. Bright Monomeric Fluorescent ...

...Gateway cloning) one cassette. Counterselection allows the scientist to select bacteria that are only resistant...plasmid-containing bacteria when the cells are grown on selective media, and provides those bacteria with a pressure...negative bacteria. In future posts, we'll detail selection in non-bacterial cells such as yeast or mammalian...resistance cassette for the mutation and for plasmid selection. Additionally, scientists can use the loss of ...

...tissue-specificity, high efficiency, and can be selected to be either integrating or non-integrating. Read...cargo of your choice. First, a viral vector is selected to perform the transduction with: either retrovirus...integrated can be cloned into the vector that you have selected. The nucleic acid will be flanked by viral packaging...and popular viral vectors. Check out our viral selections to see if we have one that might work for your...available to choose from. And remember, we have a selection of ready-made viruses as well. Transduce on! ...