...If you're just getting started, check out our Introduction to PCR.) But if you're ready to go beyond the...tips on probe chemistries, primer optimization, controls, and analysis.  As a rule of thumb, you can assume...experimental parameters (neighbor stacking energies, loop entropy effects, cation concentrations and a temperature...side and a 5% negative error on the other will introduce unwanted variation into your data. Try different...protocol may have to be re-optimized.  While troublesome, multiplexing your reference gene set can save...the fold change of each sample compared to your control. But as we said previously, just because it’s used...assumes the amplification kinetics of the target and control genes are fairly equal, an assumption that is not...

...tail vein or retro-orbitally Gauge: 27–30 Volume: < 0.2 ml Skill required: beginner (retro-orbital) to ...restrainer or anesthesia Tail vein or retro-orbital 27–30 < 0.2 mL Beginner (retro-orbital) to intermediate (tail...2020). A retrospective analysis for different routes of administration in Mice-Percutaneous Retro-Orbital...vein injections.” However, they can also be given retro-orbitally, behind the eye. IV injections are used...needle in the correct vein without collapsing it. Retro-orbital injections are also thought to be less stressful...decision Hopefully this post has provided a good introduction to common injection routes in mice! When considering...

...recombineering machinery are first electroporated with pCas9. Then, pCRISPR is introduced along with an oligonucleotide... in a bacterial strain introduced to the population as methods of introduction. Engineering the bee gut... of knockdown and developed new, more tightly controlled, Tet responsive promoters to avoid leaky activation...systems differ on how exactly the CRISPRi locus is introduced (Tn7 in Gram-positives and ICEbs1 in Gram-negatives...CRISPR system targets these genomic locations, and introduces double strand breaks, the bacteria are unable...This approach provides a more granular level of control where individual bases can be modified. Cytidine... that could be fused to dCas9 and efficiently introduce cytosine-to-thymine substitutions in target sequences...

...super-resolution microscopy or electron microscopy. Since its introduction in 2015 by the Boyden Lab, ExM...expand evenly in all directions, aka isotropically. But achieving isotropic expansion depends on how well the...2010). How do you overcome this resolution limit? Electron Microscopy (EM) is one option, but the equipment...much to be done about that.  Second, expansion introduces distortions. The goal in expansion is for your...variation, your samples can tear or expand unevenly, introducing distortions in your final images. It is possible...a deeper understanding of neuronal synapses, centrosomes, parasites, and more (Bertiaux, et al. 2021; ...resources on the Addgene blog Antibodies 101: Introduction to Immunofluorescence  Antibodies 101: Multiplex...

...protein. With this control set, you should see your target protein IP’d in the positive control sample but not... Basics of Western Immunoblotting.) For mass spectrometry, elute in a urea-containing lysis buffer. Alternatively...permanently altering the structure of the protein.  Controls As with any experiment, it is critical to include...include proper positive and negative controls for an IP. When possible, perform the IP in parallel with ...interest and a sample that does not. Common negative controls include knockout cell lines or tissues that do... not in the negative control, confirming that the capture antibody is functioning as expected. In order...perform parallel IPs with beads only and an isotype control antibody. Alternative applications Oftentimes, ...

...1998). Regulatory elements within the ori also control copy number and stability, which influence the ...which consist of a plasmid’s ori and all of its control elements, from broad host range plasmids can be...Hypomycrobium X, Legionella pneumophila, Methylophilus methyltrophus, Methylococcus methanolicus, Methylosinus trichosporium...hydrophila, Agrobacterium tumefaciens, Alcaligenes eutrophus, Azotobacter vinelandii, Brevibacterium methylicum...Klebsiella aerogenes, K. pneumoniae, Methylophilus methylotrophus, Moraxella spp., Mycobacterium aurum, M. smegmatis...Agrobacterium tumefaciens, A. rhizogenes, Alcaligenes eutrophus, Enterobacter sp., Erwinia amylovora, E. carotovora...spp., Legionella pneumophila, Methylophilus methylotrophus, Myxococcus virescens, M. xanthus, Proteus ...

...type of edit you need to introduce highly specific? Perhaps you need to introduce a single nucleotide modification...McDade and significantly updated in 2022 by Susanna Stroik.  The advent of CRISPR/Cas9 has made it easier ...right for your unique experiment. Here, we will introduce you to the wide array of Cas9s (many available...Knock-out cells or animals are engineered by introducing Cas9-mediated frameshift mutations in the gene...interest. The genomic target for Cas9 and the introduction of this mutation can be any site early in the...and knockouts Some experiments may require the introduction of a mutation at a specific site e.g., deleting...Synergistic Activation Mediator complex, generally the strongest activator, consists of a dCas9-VP64 fusion and...

...your cell of interest: AAV tropism and route of administration AAV tropism is defined by the viral capsid...FRT, with AAV vectors are common approaches to control transgene expression by restricting expression ...regulatory elements such as promoters and enhancers, multicistronic vectors, and post-translational regulatory ...Combinatorial strategies can also be used for retrograde tracing: visualizing neurons that received information...region. In the example shown in Figure 1, injected retrograde AAVs at one location result in opsin expression...viral vector related blog posts Learn more about retrograde AAVs Resources on Addgene.org Read our AAV ...guide Learn more about our viral service Find retrograde AAVs at Addgene ...

..., that strongly binds the glycoprotein, avidin or a similar protein, streptavidin. This strong interaction...antibody based affinity purification and mass spectrometry approaches. Recently however, scientists have...classic antibody based affinity purification/mass spectrometry methods.  BioID: a promiscuous biotin ligase...ascorbate peroxidase (APEX), used as a genetic tag for electron microscopy, could be used for efficient proximity...of hydrogen peroxide. This radical reacts with electron-rich amino acids such as tyrosine on neighboring...exogenous biotin allowing for more precise temporal control.  Find plasmids for TurboID and miniturbo Split-BioID...Stephanie S., et al. "Directed evolution of APEX2 for electron microscopy and proximity labeling." Nature methods...

...video making.   Cycle One: The Ironing Board Control Center With the pandemic in full swing and everyone...Ironing boards. Good as a WFH desk, and as a video control center.   Cycle 2: Zooming Forward Design: ...me to set up a camera in a room, but still have control over focus, zoom, stop/start record, and allow ...focus system. Follow focus systems allow you to control the focus or zoom on a camera lens without having...door (connected via bluetooth to the camera, to control some basic functions, including starting and stopping... complete with wireless video receiver, camera control, and extra hand sanitizer.    Cycle 4: Venturing...mount a battery to it if necessary. I could still control the camera from a distance if I needed to be close...

...updated Nov 3, 2022 by Susanna Stroik. You’ve designed your gRNA and introduced it into your target cells ... of your target site is. If you are trying to introduce a homozygous mutation, it’s helpful to know how...knockout cell line is desired, any mutation that introduces a frameshift in the coding region of interest... sequence clones to verify that the deletions introduced are out of frame but finding clones with deletions....   Validating knock-in mutations You’ve introduced a donor template along with your gRNA and Cas9...your desired edit is. Large knock-ins Are you introducing a large insertion, something 20 base pairs or...restriction enzyme sites? Never fear! You can introduce a silent, passenger edit to serve as a proxy for...

...-phosphorylated or ‘wild type’ control protein. The ‘wild type’ control can be made one of two ways. First... model systems coupled with advances in mass spectrometry suggest that phosphorylation is far more prevalent...non-phosphorylated recombinant protein and perform in vitro kinase reactions. Often, however, researchers lack...or have difficulties with kinase specificity in vitro. The establishment of a system in which purified...-containing GFP that can be used as a positive control during optimization experiments. This version of... pSer incorporation can also done using mass spectrometry to identify a mass shift proportional to a single...expression plasmid. This is a particularly important control when creating a phosphoserine-incorporation mutant...

...with the lentivirus HIV-1. Today, HIV-1 can be controlled with antiviral therapies such that the virus ... dCas9-SAM to activate HIV-1 transcription and destroy infected cells, the other using wild-type Cas9 ...Read on to learn how CRISPR can take on HIV-1 in vitro, and what obstacles must be overcome for clinical...infections not usually seen in healthy individuals. Antiretroviral therapy (ART) can virtually eliminate plasma...Why can’t the body mount an immune response to destroy these reservoirs? Essentially, the immune system...out of the genome with CRISPR/Cas9 Before the introduction of CRISPR, HIV-1 genome editing with ZFNs and...infection period rather than the ART-induced viral control found in most HIV-1 patients. Obstacles for CRISPR...

...26973 AAVrg pAAV-hSyn-hChR2(H134R)-EYFP   Control AAV Control AAV allow researchers to not only interpret...inventory. Our new AAVs include: Optogenetics AAV Control AAV Biosensor AAV And more! Optogenetics Optogenetics...more efficient tools for light mediated neuronal control and we are expanding our viral service to make ...own experiments. To this end, we offer specific control AAV in smaller aliquots of 20 ul for serotype testing... service has now expanded to offer many of our control plasmids in new serotypes. See our Serotype Testing...response to action potentials, is now offered under control of the CAG promoter for ubiquitous expression. ... 44362  AAV1 pAAV-hSyn-DIO-hM4D(Gi)-mCherry Controls Plasmid Serotype Name 50459 AAV1 pAAV-hSyn-DIO-mCherry...

...creative commons license.    The introduction The introduction serves two purposes. First, it should... to have conflict. There are a lot of ways to introduce conflict into your work, but one of my favorite... two other things to complete your story: an introduction, and a resolution.   Fig. 2: The structure...ever thought about how X works?”, or even by introducing a person that the audience can relate to. It ...read (or listen or watch) further.  Second, the introduction needs to provide enough information so that ...thing applies for science storytelling. You can introduce your results right at the beginning of your communication...Addgene blog SSCS: Cognitive Load SSCS: Preprints Intro to our Summer SciComm Series  ...

...which introduces functional Cas9 protein and the targeting guide RNA into cells by electroporation. Results...of the nuclease dissociates very slowly after introducing a cut, with residence times on DNA of approximately...free to anneal to complementary ssDNA molecules in vitro. Figure 1: Cas9 binding to the cleaved DNA... if we could leverage this model (built from in vitro studies) to boost the efficiency of sequence replacement...enables sequence replacement without cutting Our in vitro results also demonstrated that catalytically inactive...advantage but error-prone repair of the gene would be disastrous (i.e. if breaking the gene is worse than leaving...homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery." Elife 3 (2015...

...oviductal lumen, tweezer-type electrodes are placed to mediate electroporation. Ohtsuka et al. compared traditional... homology arms and a selection marker, then electroporate it into mouse embryonic stem cells. After selecting...from Yang et al. requires two gRNAs targeting two intronic regions and two repair templates in order to insert...promoter upstream of the left homology arm, perform in vitro transcription, and use the IVT product as a template...compatible with standard microinjection and electroporation methods using in mouse transgenics. Designing... allele requires you to target 2 loxP sites to intronic regions 0.5 - 0.8 kb apart. In the example below...between the ampulla and infundibulum. In vivo electroporation is performed immediately after injection. Image...

... of biogenesis and mitochondrial degradation in vitro and in vivo. Merged green and red channel fluorescence...Technology Austria, decided to take a novel approach to control for these effects, drawing on the rapidly growing...1):39-48. APEX2 for proteomic mapping and electron microscopy A thorough understanding of complex...enabled progress on both fronts – as a genetic electron microscopy (EM) tag providing superior subcellular...also available at Addgene and can be used as a control for your experiments. For more information, please... allowing labs to precisely and non-invasively control neuronal signaling. For more information on DREADDs...Chen and colleagues for genome editing inXenopus tropicalis. (Guo et al., Development. 2014..) A new, higher...

...Lab and the Chang Lab have created methods that introduce diversification by targeting a site of interest...which limits the level of variation that can be introduced.   Diversification is limited due to the integration...use of such libraries means that the diversity introduced by lambda red recombineering and Multiplex Automated...PCR, EvolvR uses an error prone polymerase to introduce mutations. However, by using nCas9 to direct PolI3m...the 3' end of the nick (right), occasionally introducing mutations (red nucleotide). Created with BioRender.com...EvolvR, three additional amino acid changes were introduced to nCas9. These changes promote nCas9’s dissociation...increased the likelihood that a single gRNA would introduce more than one mutation near the target site. Applications...

...one type of virus to choose from (lentivirus, retrovirus, AAV, etc.). When you’ve decided on an application...experiment. Research applications Gene expression control Researchers are always wanting to manipulate gene...complementary mRNA sequence for degradation. Viral introduction of shRNA plasmids can induce gene-specific silencing...Viral CRISPR-Cas9 tools have been engineered to introduce frameshift mutations in genes of interest, turning...depending on the virus used. All of these expression control mechanisms require a specific modification of the...that the copies of library cargo delivered are controllable. For many screening applications, it is essential...attenuated, nef-deleted HIV-1 strains in vivo. Retrovirology. 2007 Sep 23;4:66. doi: 10.1186/1742-4690-4-...