...interface upon thaw (Fig. 2). Before thawing, all the samples were at the control pH of 7.4 as indicated by ...showed that AAV infectivity was not altered by a short 2-hour exposure to pH as low as 2.5 (Potter et al., ...Methods. 2013 Apr;10(4):278-9. PubMed PMID: 23538862. 2. Potter M, Lins B, Mietzsch M, Heilbronn R, Van Vliet...affected the pH of our viral samples. Read on to learn how aqueous samples might be affected by dry ice...Measuring the pH of our samples: What we did To measure the pH of our samples, we used a solution of cell...when the samples were frozen. This was clear by the gradual change in pH visible at the sample interface...at -80°C. For samples that are shipped on dry ice, this can affect the pH of the sample, which may have...

...domains used in SAM. This method has been shown to activate genes at 2- to 3-fold higher than SAM, SunTag, and...activation. Synergistic Activation Mediator (SAM) Description SAM uses specially engineered sgRNAs to increase...re-engineer components of Cas systems themselves. SunTag, SAM, and VPR have all shown significant improvements ... p65). Performance When targeting single genes, SAM consistently shows the highest levels of gene activation...regulation (activating multiple genes at once), however, SAM exhibits activation levels comparable to other popular...activators while showing lower activation levels than SAM. One drawback of this method is its construction:...VP64 activator, but lower levels compared to the SAM system. Its activation levels are similar to that...

...CRISPR/Cas9 Synergistic Activation Mediator or SAM. SAM is an engineered protein complex for the transcriptional...VP64 fusion; 2. A sgRNA incorporating two MS2 RNA aptamers at the tetraloop and stem-loop 2; 3. The MS2...not been achieved.” That is, until now. Enter the SAM pooled library In a paper published in Nature in ...plasmid which expresses the activation helper protein. SAM can be combined with a human genome-wide library ...Nature paper reports, “Here we have shown that the SAM system is robust, specific, and can facilitate genome-scale...combined with a compact pooled sgRNA library. Our SAM-mediated screens exhibited a high degree of consistency...References & Further Reading: For more information about SAM, including a tool for getting optimized guide sequences...

...extraction protocol for more details.   Figure 2: An uncut plasmid is expected to form 3 bands: nicked...replicate.   How can I prevent recombination in my samples? Plasmid recombination can affect viral vectors...stable E. coli reduce the rate of recombination in samples. For a more detailed comparison of these cloning... easiest and clearest ways to determine if your sample has recombined. Choose a restriction enzyme that...

...that numbers are not switched around or misread.  2. Track the container - It is just as important to ...Every plasmid sample enters Addgene the same way. A package is delivered by a mail courier and then the.... Some samples are submitted as bacterial colonies in petri dishes, but close to 80% of samples are received...every sample can be tracked easily at each step in the storage process. After the original sample is barcoded...scanned to verify that it is the correct sample. Tracking samples - it's all about location, location, location...these samples have the 2D barcodes mentioned above. These barcodes identify what type of sample is contained... on the sample type. It is critical for us to know which plate, rack, and freezer each sample is located...

...strategies for CRISPR-HIV therapeutics - one using dCas9-SAM to activate HIV-1 transcription and destroy infected...realistic with the advent of CRISPR. Using CRISPR/Cas9 SAM to "shock and kill" To “shock and kill” the HIV-...the CRISPR/Cas9 Synergistic Activation Mediators (SAM) system available from Addgene. This system uses ...genome, which acts as a promoter, and found that SAM targeting of the enhancer region near the NF-KB binding...latent microglial line, treatment with CRISPR/Cas9 SAM increased the percentage of cells with activated ...that can produce HIV-1 toxic proteins, CRISPR/Cas9 SAM caused apoptosis, indicating a true “shock and kill...CRISPR/gRNA-directed synergistic activation mediator (SAM) induces specific, persistent and robust reactivation...

...Check out Addgene's Lentiviral dCas9 SAM vector and Piggybac dCas9 SAM vector! On/off control all the time... the transcriptional level.     Fig. 2 Tetracycline on/off promoters and modes of action ...

...library substantially identified more hits than the SAM library approach in a comparison study screening ...screening for vemurafenib resistance in A375 cells. The SAM system (CRISPR/Cas9 Synergistic Activation Mediator... PMID: 25184501. PubMed Central PMCID: MC4262738. 2. Doench, John G., et al. "Optimized sgRNA design to... in the depletion of essential genes. In the subsampling analysis of the sgRNAs, Brunello even with only...

...include Cas9 fused to VP64, VP64 along with other SAM factors, and the Suntag array of factors, all targeted...sequence is unique compared to the rest of the genome.  2) The target is present immediately upstream of a Protospacer...utilized allowing for PAM flexibility. Fig. 2: Prime editor engaging target DNA. The reverse transcriptase...recruitment of multiple activation domains to the same genetic locus via co-expression of an epitope tagged...

...the image can be acquired more quickly. 2-Photon Microscopy 2-Photon Microscopy attempts to solve two ...Fluorescence microscopy. Nature methods 2, 910-919 (2005). PubMed PMID: 16299476. 2. Conchello JA, Lichtman JW. Optical...for a new sample I always start by asking two questions: 1) Is the sample dynamic or static? 2) Is the ...collects fluorescence. Like 2-photon microscopy, only a single focal plane of the sample is excited at one time...provide a 2-10 fold increase in resolution, down to 10s of nanometers. Thick dynamic samples Ex: 3D cell...enhance spatial contrast (i.e. optical sectioning (2) or super-resolution (3). Fluorescence microscopy...photobleaching and a reduction in signal intensity. 2-Photon microscopes restrict excitation (and bleaching...

...previously developed method to detect SARS-CoV-2 in the samples. First, RNA is extracted and subject to reverse... State University. Since its appearance, SARS-CoV-2 has spread to almost every part of the world manifesting...Rapid detection of 2019 novel coronavirus SARS-CoV-2 using a CRISPR based DETECTR assay Sensitivity:10-...primers that target the E and the N2 gene of SARS-CoV-2. If the Cas12a-sgRNA complex binds to the target sequences... method has been optimized to detect the SARS-CoV-2 virus directly using saliva without having to perform...and visual detection of novel coronavirus SARS-CoV-2 Sensitivity: 4.6-11 copies/microliter of RNA targets...single-molecule sensitive. In the case of HIV-1 and SARS-CoV-2 detection, AIOD-CRISPR without preamplification was...

...Illustration of SARS-CoV-2 from Philippa Steinberg for the IGI.   What SARS-CoV-2 plasmids from Ginkgo ...Bioworks to synthesize and share over 1,850 SARS-CoV-2 plasmids to help academic and industrial labs accelerate...request? Currently, plasmids containing all SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...bacterial expression systems, Ginkgo synthesized SARS-CoV-2 expression plasmids in a high copy backbone with a...high level expression and purification of SARS-CoV-2 proteins for downstream experiments and offer flexibility...optimized, full length and truncated versions of SARS-CoV-2 S (spike protein) cloned into a CMV driven expression...first deposited set of plasmids containing SARS-CoV-2 protein coding genes in E. coli and S. cerevisiae ...

...loading control in the same lane, and the resulting fraction is reported. Figure 2: Normalization with...condition 1” than “condition 2.” By eye, the loading control appears about the same between the two conditions... than the analogous green “condition 2” points by the exact same value. For the two solid POI points, ... of your POI as a fraction of the total sample. Assumption #2: The loading control can be accurately quantified...control band is weaker in condition 1 than condition 2, the normalized values for the two conditions are ...opposite change in your loading control. Assumption #2: The loading control can be accurately quantified ...consider a different loading control. Assumption #2: The loading control can be accurately quantified ...

...repository of tools for SAR-CoV-2 research. Two new antibodies, Anti-SARS-CoV-2 Nucleocapsid Protein [mBG17...Anti-SARS-CoV-2 Nucleocapsid Protein [mBG86], target distinct epitopes on the SARS-CoV-2 nucelocapsid ...Anti-SARS-CoV-2 Antibodies here! Terry, J.S., et al. (2021). Development of a SARS-CoV-2 nucleocapsid ...Anti-SARS-CoV-2 Nucleocapsid Protein antibodies #211756 (left panel) or #211757 (middle panel). Samples were also...prime editor PE7 Antibodies to facilitate SARS-CoV-2 research New viral vector preps of Voltron2 voltage... incorporates La’s N-terminal domain, PE7 (Figure 2).  PE7 shows enhanced editing efficiencies in a number...editor. Find PE7 plasmids here!     Figure 2: PE7 improves prime editing. A) Schematic of PE7 prime...

...fragments that retain antigen binding (Figure 2):  F(ab')2 - These fragments are produced by proteolytic...fragments are produced by mild reduction of F(ab')2 fragments, which reduces the hinge region disulfide...Variable (scFv). Size: ~25 kDa.   Figure 2: Comparison between a full IgG antibody and common...advantages, though with some caveats.  First off, F(ab’)2 fragments are bivalent, so may have higher avidity...or Fab fragments. Higher avidity means that F(ab’)2 fragments may have overall stronger interaction with...Clinical Prospects of Antibody Fragments. Antibodies 8(2):28. https://doi.org/10.3390/antib8020028    Wessel...multiplexed super-resolution microscopy. Cell Rep Methods 2(10):100301. https://doi.org/10.1016/j.crmeth.2022.100301...

... step of the series dilutes by 1:2 for a series that ranges from 1:2–1:32. The specific dilution series...series ranging from 1:2 to 1:32. Each individual step of the series is consistent at 1:2. When preparing a...of the series diluted by the same factor. For example, Figure 1 depicts a 2-fold dilution series. Each ...the unknown sample based on its measured response. An example of this is shown in Figure 2.   ...accuracy of the curve, avoid pipetting small volumes (< 2 µL) or volumes too large for a standard micropipette... Figure 2: The concentration of a serially diluted standard is plotted against its OD560 measurements...molecule in an unknown sample. To do this, you serially dilute a control sample of known concentration...

...recruits transcriptional activators such as VP64, SAM, or SunTag to specific ncRNA loci to enhance or upregulate...You’ve probably heard that only 2% of our genome is made of protein-coding genes, and you might be wondering...insertions or deletions in the DNA sequence. This same approach is effective to target short ncRNAs, such...useful for targeting thousands of long ncRNAs at the same time (Liu et al. 2018). However, removing large ...

...status of MEK1/2 was tracked at 5 minute intervals via Western blot. Phosphorylation of MEK1/2 peaks at just... phosphorylated MEK1/2 levels, suggesting PP1/PP2A is responsible for this MEK1/2 negative feedback. ...PubMed PMID: 28232577. Figures 2-4 were modified from this reference. 2. Regot, Sergi, Jacob J. Hughey...precisely control the timing of kinase activity. 2. Studying negative feedback loops of signaling pathways... studied the negative feedback on endogenous MEK1/2 phosphorylation by creating a short pulse of psRaf1...activity. They did this by exposing psRaf1 to a short 2 minute pulse of 500 nm light (turn on psRaf1) followed...hypodermis and gut cells of C. elegans causes the same phenotype. Activation of the Raf-MEK-ERK signaling...

... the same site, is 2’-fluoro (2’-F), which substitutes a fluorine for the hydroxyl group at the 2’ position...most common of these is 2’-O-methylation, where a methyl group is added to the 2’ hydroxyl group of the...them together, for example phosphorothioates with 2’-O-methyl modifications, proved more stability than...generated by introduction of a single photocleavable 2-nitrobenzyl linker in the 20-bp targeting region of... when scientists talk about their precious RNA samples. RNA is inherently less stable than most macromolecules...CRISPR-Cas9 experiments – the “gRNA” – suffers from the same issues as your experimental RNA. To solve this problem...protein and can increase on-target efficiency. In the same vein, locked nucleic acids or bridged nucleic acids...

...   Class 1 versus class 2 There are two CRISPR classes: Class 1 and Class 2. Class 1 CRISPR systems use...systems to carry out various functions. Class 2 Class 2 CRISPR systems rely on a single protein Cas effector...explore Class 2 CRISPR in more depth, the Jennifer Doudna lab maintains an encyclopedia of Class 2 CRISPR systems...systems: a burst of class 2 and derived variants. Nature Reviews Microbiology, 18(2), 67–83. https://doi.org...complex for antiviral defense) complexes, while Class 2 use single protein Cas effectors, like Cas9. The Cascade...1 remains largely underutilized compared to Class 2. Class 1 Despite their incredible abundance, Class...components present in the Cascade complex. Figure 2: Schematic of a Cascade complex (blue subunits) with...