...Workflow of screens and selections   Choosing between selections and screens Selections sound like a great...and selections The common goals The confusion in differentiating between a screen and a selection isn’...considering either a cellular screen or a selection. A selection is a process in which only cells with the...similar to enhancer/suppressor screens. Selections Selections differ from screens in that only survivable...population. Selections do require there to be a culture condition that can apply enough pressure to select for...are not conducive to selections and have to be addressed with screens. Selections require there to be ...expedition.” Your first question is: to screen or to select? Which method will work best for your scientific...

... provide you with some advice on the antibody selection process. Antibodies are not one size fits all...your application.      Fig. 1: Antibody Selection Flow Chart    What does validated mean anyway...validation is the most important factor in antibody selection, but what does that mean? Not all validation is...Antibodies 101: Validation!  Download our antibody selection flow chart.  References and Resources Additional...

...your positive selection strategy, here are a few to consider. Antibiotic selection Selection is straightforward... ways to do this involving positive selection, negative selection, and/or screening. We’re focusing on... Positive selection: Cells that have gained a specific gene survive In positive selection, only cells...of interest. In these cases, antibiotic selection will select for cells that have been transformed with...interest. Auxotrophy Besides antibiotic selection, you can select for a successful uptake of your DNA of... Negative selection: Cells that have lost a specific gene survive Unlike positive selection, negative ...negative selection means you’re selecting for the loss of a gene product - usually something toxic. This gene...

...characteristics to consider when selecting a dye or ligand to make your selection easier, as well as highlight...throughout the past century, it can be overwhelming to select the optimal dye or fluorescent ligand when designing... options without even realizing it. Yet, this selection is crucial as it will impact downstream outcomes...and efficacy of findings. If you’re struggling to select the right fluorescent tools, this blog is here ... green light emission. Key considerations in selecting fluorescent dyes/ligands Effects on protein function...consideration for assay design (Breen et al., 2016). When selecting dyes, it's important to test them under experimental...quenching, which can be mitigated through careful selection and optimization. Fluorescent experiment types...

...commonly used for positive selection. While the positive selection marker selects for recombination, the ...positive selection, but it will be sensitive to negative selection. Remove the positive selection marker...by homology arms. The negative selection marker HSV-tk is used to select against random recombinants. ...homology arms. When we select cells later, we will first perform a positive selection for the antibiotic ...resistance gene and then a counter selection for the negative selection marker - this latter step will kill...the negative selection marker selects against improper, random recombination into a different locus. Correctly...negative selection marker, but cells with random recombination may incorporate the negative selection marker...

...backbone. This step requires “counter-selection,” meaning that the selection is for the loss of a specific phenotype...discussed below).  Counter-selectable marker: sacB What are the counter-selectable markers used to identify... of toxin could select for mutations in the toxin itself (leading to counter-selection escape). The rhamnose...mutations. Modifying bacterial genomes using counter-selectable markers and homologous recombination As an alternative... because these events are rare, each requires selection. These two steps are (Figure 1): The initial...in a merodiploid. This can be accomplished by selecting for an antibiotic resistance marker from the vector...double crossovers? The most commonly used counter-selection system relies on the sacB gene product of B. subtilis...

...scientsts do not "select" for cells that have taken up the plasmid in the same way. Selection methods, described...Positive selection in mammalian cells To achieve stable transfection, there should be a selective pressure...table below we focus on positive selection; however, negative selection techniques can be used in conjunction... including how replication occurs and whether selection is necessary for transfected cells. Before getting...cell lines and are not used for general plasmid selection. Instead, reporter genes are often employed to...purposes of this post, we will define positive selection as the means of picking up positive traits (i....cells resistant to a toxin), whereas negative selection would be the picking up of a negative trait (i.e...

...means of selection in yeast cells, in addition to the bacterial ORI and antibiotic selection markers.  ...replication.  Selection markers for yeast Historically, scientists have utilized auxotrophic selection rather...references.  Yeast selection marker What does the marker help synthesize? Counterselection? (growth-based... auxotrophic markers as a means of selection: A specific selection marker needs to be used with a yeast...improvements in antibiotic selection have made utilizing the more traditional drug selection methods feasible in...rather than antibiotic selection when working with yeast, due to high rates of spontaneously occuring resistant...table below lists some of the most commonly used selection markers in yeast and provides the element needed...

...mutation co-selection. Only animals rescued for the conditional lethal mutation survive the selection; from ...enrich for editing events without the need for any selective marker to remain in the edited animal. Development... love to rely on the awesome power of genetic selection. Knock-in events tend to be rare, and the challenge...these edits. Initial work in C. elegans relied on selective markers such as drug resistance (Chen et al., ...being introduced. Cre-mediated excision of the selective cassette minimizes the sequence added, leaving...published in Genetics detailed approaches in which selection for an editing event that produces a visible phenotype...used inactivation of the unc-22 gene as their selection marker (Kim et al., 2014). Meanwhile Andy Fire...

...you to select against the presence of a transgene instead, which can’t be done if your selection method...Selectable transgene markers for plant transformation fall into two main categories: antibiotic/herbicide...herbicide resistance and fluorescent proteins. Selection using resistance genes usually involves killing ...alleles or off-target gene editing. Antibiotic selection would only then kill the desired plants! PCR or...markers are currently used in these situations to select against CRISPR positive plants, but screening these...Photos by Andrew Willoughby   Using RUBY as a selection marker The creators of RUBY highlight its use ...collection could contaminate it. I have used it for selecting transformation events and am currently planning...

... are dry, it’s time to select for transformants. It’s very important to select multiple T1 plants from...insertion in the absence of selection. For a protocol for antibiotic selection, check out Harrison et al...plants on selective plates for too long: even resistant plants will be affected by the selection eventually...for tDNA insertions into the plant’s germline Selecting for seeds that have the tDNA insertions (usually...(HA, FLAG). There are also a variety of plant selection markers (basta, hygromycin, kananamycin, tunicamycin...Dinneny lab that additionally allow for visual selection of transformants based on expression of RFP in...non-transformed seeds from the pool you will be selecting from later. The Arabidopsis transformation process...

... that demonstrates selectivity for cell surface labeling may become less selective if used in intracellular... to confirm (or refute) that the antibody is selectively detecting the target-of-interest in your assay...the approaches that can be used to determine selectivity of an antibody for flow cytometry experiments...well as a pragmatic approach to determining the selectivity of antibodies for other sample types and for ...For these reasons, it is essential to test the selectivity of your antibody. But testing is not one size...proteomic and transcriptomic datasets can be used to select a candidate cell line (e.g. DepMap) – you’ll need...producing flow cytometry characterization data for selected antibodies, using the knockout approach. You can...

...within it by selecting for Zeocin resistance. Then, counter-selection with methanol medium selects for those... cloning, where the ccdB gene serves as a counterselectable marker in the destination vector. Successful...active. Shen Yang’s lab made use of mazF as a counterselectable marker to create unmarked genetic modifications...pastoris. By combining both positive and counter selection in one plasmid, they were able to create an efficient...site-directed mutations, all without introducing unwanted selection markers. Restriction modification systems Because...bioluminescence reporter in vivo in the absence of selective pressure and found that plasmids were maintained...activity over 48 hours in vitro without antibiotic selection. In a subcutaneous cancer model, the researchers...

...modifications: insertion and deletion of selectable and non-selectable sequences, point mutations or other ...including selectable DNA fragments, such as antibiotic resistance genes, as well as non-selectable DNA fragments...be used to select for positive clones but, because mini-λ stably integrates, drug selection is not required...Electroporation of substrate DNA and outgrowth of bacteria. Selection and confirmation of recombinant clones.    ...expression is that it doesn’t require antibiotic selection to maintain expression of the recombineering system...event. Alternatively, if the modified allele is selectable, it can be transferred to a different genetic...prophage is stable and no longer requires drug selection.  One drawback to this approach is that it requires...

...Jaffrey group selected for a 49-nucleotide aptamer they called Broccoli. As a product of SELEX and FACS, ...Szostak and Larry Gold, developed methods for selecting and amplifying RNA sequences that could bind very...evolution of ligands by exponential enrichment (SELEX), some 1010 oligonucleotides could be screened for...aptamers, from the Latin aptus, meaning “to fit”. SELEX could be used to classify DNA aptamers as well, ...antibodies and enzymes, such as: Design and selection for targets can be done largely in vitro; Easy... low fluorescence in vivo, the Jaffrey lab used SELEX to find a 98 nt RNA aptamer that would bind the ...biosensor possibilities. For instance, further selection of Spinach with a target metabolite creates an...

...and multiple selectable markers. Bob Goldstein’s lab previously used a transgene selection strategy for...applications, SEC selection could replace other, more restrictive C. elegans selectable marker systems. ...hours worth of hands-on time. Creating a modular, selection-based gene tagging approach Multiple features ...well as a second injection step to remove the selection marker. Other labs have used PCR-based screening...Dickinson et al. sought to create a new and efficient selection-based CRISPR/Cas9 method to generate knock-ins...any genetic background. Their new method uses a selection cassette containing genes for hygromycin resistance...laid eggs, hygromycin is added to the plates to select for candidate knock-ins. These candidate animals...

...should be used in conjunction with selection methods. Combining selection and screening ensures that the ... way to select for plasmids containing an insert, there are other methods. Positive selection vectors ...posts here. Now that we have covered antibiotic selection here at Plasmids 101, we can talk about an even... screening your cloning reaction. Being able to select for colonies that contain your plasmid is a great...that contain plasmid with an insert? Blue-white selection is a widely used method to do just that! (To read...is just that – a screening process. It does not select only those cells that have taken up a plasmid and...disruption in the blue-white screen. Antibiotic selection is also used in conjunction with this method to...

...template to facilitate the selection of cells containing your desired edit. The selection is marker is then seamlessly...method of making stable cell lines is to transfect, select, and isolate clones. This procedure is cumbersome...transgene). To overcome this obstacle, a positive selection cassette can be incorporated, and used during ...Simply put, the piggyBac™ transposon containing a selection marker is included in a standard homology directed...By using a donor plasmid containing a negative selection marker such as thymidine kinase, a researcher ...2: Editing DNA using the piggyBac™ transposon selection technology using a site-specific nuclease to stimulate...recombination and Footprint-Free™ removal of the selection cassette by excision-only piggyBac™. Image courtesy...

... 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...

...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...