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...activation. Synergistic Activation Mediator (SAM) Description SAM uses specially engineered sgRNAs to increase...domains used in SAM. This method has been shown to activate genes at 2- to 3-fold higher than SAM, SunTag, and...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...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... sam pooled library...

...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...

...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...therefore particular sample is missing so that we can make a new tube from a replicate sample. The transformation...

...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...

...  Addgene’s old homepage had been relatively the same for quite a while and hadn’t kept pace with our ...better ways, we realized that Blugene needed the same thoughtful treatment. Blugene has been with us since...

...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...describing how short term storage on dry ice can shift sample pH, we wondered whether or not the dry ice we use...frozen during shipment was having an impact on the samples. We therefore devised a few experiments to determine...the tube, the CO2 can alter the pH of the liquid sample. The good news is that this effect is reversible...

...Novophage to Sample 6. To learn more about Novophage's successful transformation into Sample 6 and Dr. ... in food products prior to Sample 6 16:27 - 18:02 : Benefits of the Sample 6 technology and how It works...- 19:56 : Why Sample 6's first test was for Lysteria 19:56 - 24:41 : Who is using Sample 6 and why? 24...Resources Visit the Sample 6 website Check out Dr. Koeris' Website, biotechstart.org Read Sample 6's Paper on...for use in a biotech startup 12:16 - 15:15 : Why Sample 6 began to develop tests for food products as opposed...Directors. We're excited to watch both Addgene and Sample 6 grow with his help! Special thanks to our sound... sample6...

...Check out Addgene's Lentiviral dCas9 SAM vector and Piggybac dCas9 SAM vector! On/off control all the time...

...JohnInnesCentre rule pic.twitter.com/WMb97fpUOI— Sam Mugford (@samtmugford) December 3, 2021   Congratulations to...

...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... in the depletion of essential genes. In the subsampling analysis of the sgRNAs, Brunello even with only...

...can use for your experiments. We have found that SAM (Konermann et al., 2014), Suntag (Tanenbaum et al..., check out our blog post covering VP64, Suntag, SAM, and VPR. Worry less about off targets Unlike Cas9...

...proteins in tissues, cells, and biofluids. Recently, Sam Myers, a Scientist in the group, developed a CRISPR-based...and RNA sequencing of complex heterogeneous RNA samples. Furthermore, such high processivity enzymes and...

...RNAs Yong-Sam Kim’s lab also created a miniature yet active Cas system starting with the same Un1Cas12f1...

...recruits transcriptional activators such as VP64, SAM, or SunTag to specific ncRNA loci to enhance or upregulate...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 ...

...include Cas9 fused to VP64, VP64 along with other SAM factors, and the Suntag array of factors, all targeted...recruitment of multiple activation domains to the same genetic locus via co-expression of an epitope tagged...

...microscope to use for a new sample I always start by asking two questions: 1) Is the sample dynamic or static? ...? 2) Is the sample thin (< 15 μm) or thick? These questions will place each sample into one of four categories...capture in these types of samples is their fast movements. Depending on the sample, a temporal resolution... fluorophores higher in the sample. TIRF is also very gentle on the sample as the majority of the laser...static samples Ex: Fixed tissue sections (> 15 um), 3D cultures, and cleared tissues These samples always...onto a camera for visualization. Illuminating a sample in the opposite direction to fluorescence collection...plane within a relatively thicker three dimensional sample (see Figure 1); similar to an MRI or CT scanner...

... replicates are repeated measurements of the same sample and are included to assess variability in the... the tau western blot several times with the same samples and compare the results across runs. If the ... wild-type and knockout samples. In this test, wild-type and knockout samples are treated with the identical...present in the wild-type samples and absent in the knockout.  If knockout samples are unavailable, try substituting...interactions between the sample and the secondary antibody. C, a label control treats the sample to the full protocol...interpretation of results. A positive control is generally a sample or group that will have a desired response. For... tau hyperphosphorylation. You could run brain samples from both groups on a western blot and look for...

... easy to measure sample concentration and purity using only a tiny bit of the sample itself — so easy ...instrument can calculate a sample’s nucleic acid concentration based on the sample’s absorption at 260 nm (...spectra of a mixed sample.  But, other molecules that may be present in the sample also absorb light...levels exceed 70% of the sample content (Loughrey and Matlock, 2016). So, even samples with “good” A260/280...nm, you may have phenol in your sample.  RNA contaminants DNA samples can also be contaminated with RNA...especially problematic in low-concentration samples (samples with concentrations < 10 ng/μL), where absorbance... how much plasmid DNA you have or how pure your sample really is?  To assess the concentration and purity...

...molecule in an unknown sample. To do this, you serially dilute a control sample of known concentration...standard, standard dilutions, and unknown sample(s) in the same buffer if possible, or very similar buffers...their bicinchoninic acid assay (BCA) to normalize samples before running a western blot. Standard curves ... to extrapolate the concentration of an unknown sample. For example, a BCA assay uses a serial dilution...control protein, incubated alongside an unknown sample with the BCA reagent. The absorbance of both is...you to determine the concentration of the unknown sample.  How do I make the standards for a standard curve...standard curve by serially diluting a known control sample, called a standard. The ideal standard curve has...