...specificity control, secondary antibody specificity control, label control, and cross-reactivity control when...helpful to focus on something controllable, like experiments (and their controls!). This blog post will discuss... ins and outs of controls for biological experiments, starting with general controls and then moving on... of controls and be able to use this information to design thoughtfully and thoroughly controlled experiments...experiments.  Broadly useful controls Positive and negative controls Most biological experiments require...positive and negative controls to ensure proper interpretation of results. A positive control is generally a ...serum or isotype-specific control (Hewitt 2014, Alexander 2018). In this control, the primary antibody is...

...replicate controls.  Transfection controls: empty vector and internal control A transfection control measures...Negative controls: untreated cells, empty vector control, and non-targeting control Negative control conditions...lacking the proper controls are invariably inconclusive and unreliable. Proper controls provide the constant...about the various controls that can be used for plasmid-based experiments. What are control plasmids? Generally...experiment, delineate its requisite control plasmids, and discuss why these control plasmids are critical for the...use of proper control plasmids would address and answer these questions. Types of control plasmids Part...the target cells. One transfection control is an empty vector control; specifically, the plasmid without...

...loading control is not perturbed by the experiment The loading control is supposed to control for, well...western blots benefit from a positive control — a loading control — that you know is present in every lane...transfer. Figure 1: A loading control is a type of positive control necessary to demonstrate a POI’s...loading control: the intensity of each POI band is divided by the intensity of the loading control in the...with loading controls. Under each lane, the fraction of the POI band over the loading control is shown. ...loading control. First, let’s explore why these assumptions are key to using loading controls accurately...opposite change in your loading control. Assumption #2: The loading control can be accurately quantified ...

...familiar with the basics of controls in experimental design, you’ll need a few controls specific to flow as an...nonspecific binding. Flow specific controls The three types of flow cytometry controls we’ll discuss are single ... may need to include additional controls like FMO or isotype controls, which we’ll describe a bit later...color control for every fluorophore you intend to use in your panel. Using single color controls On a ...your single color controls may not be accurate for your samples. The single color controls should be used...plots for a single color control. A) A flow plot showing a single color control for the APC channel. B)...likely to require FMOs. Isotypes Isotype controls are negative controls that allow you to determine what level...

... What kind of control? On, off, tunable, and/or inducible? How long do you need control? Will temporary...temporary control (a few days) be enough, or do you want a permanent control mechanism? Where is the control...vector! On/off control all the time If your experimental system requires tunable control - or maybe you...forever? You don’t need to resort to the force to control protein expression in mammalian cells: easy to ... will review tried-and-true protein expression control tools as well as explore some new innovations. ...temporarily, we’ve got you covered. Defining your control requirements To pick the best tool for the job,...protein expression. If you need the ability to control when you turn off expression for good, the Cre-...

...DNA.  Plasmids are said to be under stringent control when they depend on the host cell’s initiation ...replication independently are said to be under relaxed control, as they only require the host's replication machinery...replication mechanism of plasmids under stringent control, such as pSC101. DNA replication and regulation...round of replication. Initiation is the primary control point of DNA replication in bacteria, so the process... the bacterial chromosome and for stringently controlled plasmids. Binding of IHF (green) redistributes...binding is necessary to initiate replication, controlling the amount of active DnaA available is one of...source, ATP binding is one more way for the cell to control DNA replication. A variety of other proteins also...

...light-based control of signaling pathways. This is particularly exciting because using light to control signaling...  Option #2 may sound like fiction, but light-controlled protein purification is now a reality thanks ..., and their use for protein purification and controlling cell signaling. Key components of OptoNBs OptoNBs...insertion into loops 1 and 6 resulted in light controlled binding, but with opposite effects: insertion...insertion site on light-induced binding.  Light controlled protein purification with OptoNBs The first application...application for OptoNBs takes us in vitro: light-controlled affinity purification of unmodified proteins....stuck to the surface of a bead.  Using OptoNBs to control cell signaling The second application for OptoNBs...

...viral prep pAAV-Ef1a-fDIO EYFP AAV5. AAV8, AAVrg Control Karl Deisseroth New serotype (multiple) rAAV-CAG...pAAV-AiE0452h-minBG-SYFP2-WPRE3-BGHpA (Alias: CN2237) AAV-PHPeB Control Jonathan Ting New viral prep AiP12787 - pAAV-...minBG-SYFP2-WPRE3-BGHpA (Alias: CN2787) AAV-PHPeB Control Jonathan Ting New viral prep AiP12610 - pAAV-...pAAV-AiE0780m-minBG-SYFP2-WPRE3-BGHpA (Alias: CN2610) AAV-PHPeB Control Jonathan Ting New viral prep AiP13755: pAAV-AiE0743m...10aa-H2B-WPRE3-BGHpA (Alias: CN4496) AAV-PHPeB Control Jonathan Ting New viral prep AiP14825: pAAV-AiE0441h...prep pAAV_BiSSTe10_dTomato_nlsdTomato AAV-PHPeB Control Gordon Fishell New viral prep pAAV_BiSSTe10_ChR2... pAAV_BiCHATe27_dTomato_nlsdTomato AAV-PHPeB Control Gordon Fishell New viral prep pAAV_BiCHATe27_...

...Noninvasive Controlling Stimulation ExogenousEasily controlled EndogenousNot easily controlled Unique...spatiotemporal control of a small subset of neurons is necessary, use optogenetics. If broad control of whole...boundaries in neuroscience by granting targeted control over neuronal activity. While they serve similar...optogenetics differ are: Timing Targeted manipulation Controlling stimulation Invasiveness  Choosing the best ...Chemogenetics does not offer the same level of control over timing. Because chemogenetics relies on chemical... can reach target cells throughout the body. Controlling Stimulation Some studies rely on delivering reversible...stimulation to receptors. Optogenetics offers better control over these factors as the light source can be easily...

...concepts discussed in the paper. The transcriptional control of multiple loci is deftly coordinated by the eukaryotic...tinkering with regulatory networks and multiplex gene control. However, these approaches are often met with the...Modifying the CRISPRi system for multidirectional control The CRISPR interference (CRISPRi) model consists...CRISPRi system for more flexible multidirectional control, a new study repurposes the sgRNA to include modular...The use of regulators allows a greater degree of control of desired expression levels dependent upon the...of simultaneous and multidirectional multiplex control via scRNA design is readily apparent in metabolic...elegantly demonstrated the potential of this to control a metabolic network in S. cerevisiae by using three...

...is sufficient. Controls: Controls can make or break an experiment. The best controls for a colony PCR... They also serve as controls for your PCR reaction. Running a no template control PCR reaction for detecting...bacterial debris. Use a positive control. A good positive control is bacteria transformed with the same...primer design. Use a negative control strain. A good negative control strain is an untransformed culture...type of control is especially important for insert-specific primers. If your negative control amplifies...number of background colonies on your no ligation control plate. The more background, the more colonies you...backbone vector with and without an insert. These controls are quick references you can use when you run ...

...an appropriate control? Many CRISPR screens use non-targeting guides as negative controls. Non-targeting...are well characterized and strategies exist to control for these faults. However, it’s still unclear if...screens and how best to design these screens to controls for flaws. Recently the Bassik Lab at Stanford...site in the genome. This means that while they control for many of the effects of gRNA and Cas9 expression...detecting Cas9 cutting. Safe targeting guides better control for non-specific toxicity than non-targeting guides... a coding region. Targeting these sites should control for the effects of guide expression and dsDNA breaks... the genome, and are commonly used as negative controls for genome-wide CRISPR screens; and 3) safe-targeting...

...tracking for instance) can be controlled with FLP-FRT. For further temporal control, Tamoxifen inducible Cre...system, but it only gives you a single level of control without modification - either Cre is there or it...for site specific (and often cell type specific) control of DNA recombination and gene expression can be...addition, a variety of means to spatiotemporally control FLP and Cre expression have been developed. Read...FLP and Cre enable additional levels of genetic control. What is FLP FRT and how does it work? FLP recombinase...expressing the light activatable ion channel under the control of Cre recombinase are used in combination with...occur at a specific time point? To allow temporal control, ligand regulated Cre and FLPe recombinase have...

...and other proteins controlled by GAL4 and ZIF268. To demonstrate independent control of gene expression...a GAL4/estradiol controlled transcription factor and a ZIF268/progesterone controlled transcription factor...transcription factor 1 controls expression of transcription factor 2, which directly controls expression of a...chemically inducible approaches to provide temporal control, this new system, Fkbp/Frb Inducible Recruitment...PubMed PMID: 28916764. Plasmids for independent control of gene expression in yeast Article contributed...yeast transcription factors that can independently control expression of your genes of interest. These transcription...possible to use synthetic transcription factors to control the level of random variation, or noise, in reporter...

...-GFP AAV9-X1.1 Controls Edward Boyden New serotype pAAV-hSyn-EGFP AAV PHP.eB Controls Bryan Roth New...CN3905) AAV PHP.eB Controls Jonathan Ting New viral prep pAAV-CaMKIIa-EGFP AAV1 Controls Bryan Roth New ...minBG-SYFP2-WPRE3-BGHpA (Alias: CN3044) AAV PHP.eB Controls Jonathan Ting New viral prep AiP13038 - pAAV-AiE0743m...minBG-SYFP2-WPRE3-BGHpA (Alias: CN3038) AAV PHP.eB Controls Jonathan Ting New viral prep AiP12408 - pAAV-AiE0600m-minBG-SYFP2...pAAV-AiE0600m-minBG-SYFP2-WPRE3-BGHpA (Alias: CN2408) AAV PHP.eB Controls Jonathan Ting New viral prep AiP13863 - pAAV-AiE0888m_C4...minBG-SYFP2-WPRE3-BGHpA (Alias: CN3863) AAV PHP.eB Controls Jonathan Ting New viral prep AiP13905 - pAAV-AiE0441h...New serotype pAAV-Ef1a-DIO mScarlet AAV5 Controls Karl Deisseroth New serotype pAAV_hSyn-PdCO-mScarlet-WPRE...

...deliver DNA encoding Cre recombinase under the control of the synapsin promoter. It can be used to drive...genes to various brain regions. You can partially control which cells are successfully infected by an AAV...serotype, but occasionally you may want to further control where your gene of interest is expressed.  A cell-type...cell-type specific Cre can provide you with that control in a modular way. For example, say you would like...contains a Cre-dependent mCherry cassette under the control of an otherwise ubiquitous promoter. You could ... make an entirely new mouse line with mCherry controlled by a neuron specific promoter or you could breed...pmSyn-EBFP-Cre AAV. Because Cre expression is controlled by the synapsin promoter, it and therefore mCherry...

...AAV PHP.eB Controls Wilson New viral vector pENN.AAV.EF1a.eGFP.WPRE.rBG AAV PHP.eB Controls Wilson New...pAAV-CaMKIIa-EGFP AAV PHP.eB Controls Roth New serotype pAAV-hDlx-Flex-dTomato-Fishell_7 AAV2 Controls Fishell New serotype...  AAV PHP.eB Controls Gradinaru New serotype pAAV-CaMKIIa-mCherry  AAV PHP.eB Controls Deisseroth New...New serotype pAAV-Ef1a-mCherry  AAV PHP.eB Controls Deisseroth New serotype Looking for more viral...

... rather blunt instrument for controlling CRISPR. To bring greater control to Acr activity as a biological...Listen to this episode of Hot Plasmids! Optogenetic control of CRISPR-Cas9 by CASANOVA Article contributed ...protein domains, they have developed a system to control CRISPR-Cas9 activity using blue light (Bubeck et...opens up the potential for spatial and temporal control in many genetic targeting and editing experiments...photostable, and is compatible with optogenetic control. The researchers also showed that Archon1 is functional... both GOIs, expression levels are more evenly controlled. The Grefen Lab has utilized the 2in1 cloning...photoswitchable protein podcast segment Reversible control of photoswitchable proteins, which change in protein...

...SARS-COV-1 plasmids. Export control license process    An export control license, once approved, allows...repository, including rabies plasmids, are export-controlled materials in the USA, where Addgene is located...applicable research project when purchasing export-controlled materials. Addgene will then apply for the license...   Due to recent regulation changes, an export control license for rabies materials may be required for...application process.  More information about export control licenses is available through the US Bureau of ...

...delivering control shRNAs, GFP, and rtTA from the Tet-on system. The first two can serve as controls in a variety...virus, and quality control data for each of the materials we distribute. Quality control info can be found...The viral preparations undergo rigorous quality control testing at Addgene meaning they come ready made...used to activate expression of a gene under the control of the tet operator. AAV AAVs are the workhorses...are already available allowing you to chemically control neuronal activity while studying your phenotype...of interest. Optogenetic tools for similarly controlling neuronal activity with light will be available...determined by the serotype. You can use these as controls to determine if your AAVs are getting to the right...