...cut-to-mutation distance"  Even though we had controlled over-active Cas9 and repeated our experiments with blocking...have sufficient on-target and very low off-target activity, but should also mediate a DSB at optimal distance... the desired zygosity. Furthermore, re-editing activity of the guide RNA should be minimized by incorporating... events were corrupted by unwanted indels on the same allele, probably due to concomitant NHEJ repair....blocking mutation and our intended mutation were on the same oligo, the intended mutation rarely got in. It seemed...mixed with another ssODN template that contains the same CRISPR/Cas-blocking mutation but not the intended...

...ways to incorporate science themes into our fun activities and Halloween is no exception. For the past 10...grasshoppers, including those linked to neurotransmitter activity and geotactic behavior (Biron et al., 2005).   ... 2015). There, it divides into two cells. At the same time, it sucks out its contents (hence the name ...

... continue to be useful for monitoring neuronal activity in the form of calcium dynamics. (For more details...fluorescence) and is useful for imaging signaling activity in large populations of densely-labeled neurons.... Likewise, a lot of scientists have requested samples of AAV for testing purposes. To address this, we... serotype testing AAV, which are small (20 ul) samples of EGFP-expressing AAV packaged in various serotypes... kit with all the serotypes, since these 20 ul samples are distributed individually. See our Serotype ...neurons because background fluorescence from inactive neurons is reduced. This variant has an approximately...

...Reproducibility for Everyone Workshop included this interactive activity during their session. Image from Reproducibility... more initiative. “You have to actively reach out. It’s more proactive versus organic networking,” she...both days. pic.twitter.com/4MysvpzwkP — Samuel Guay (@SamGuay_) June 18, 2020 Final thoughts on virtual...at once, virtual conferences may have apps or interactive calendars that you can use to help plan your ...the Q&A in the chat box. Presenting? Make it interactive! As a presenter, it can be difficult to keep ...spend with the conference. “If you want to have the same experience as an on-site conference, you really ...

...and ClaI are sensitive to methylation and their activity may be blocked. If you have to use these enzymes...bacterial strain such as JM110 or INV110. Avoid star activity. Some endonucleases (for example BamHI) are capable... The glycerol in the buffer will make sure your sample settles in the gel well and the dyes provide a ...you to interpret the bands that you get in your sample lanes. Choose your ladder based on the expected...approximate the DNA mass of comparably intense samples of similar size. Run the gel at 80-150V until you...

...near future. Science on Twitter Addgene: You are active on twitter @nicolabiologist, where you tweet about...communication tool for work. Most Sainsbury scientists are active on Twitter. Sometimes it’s a good way to find out...happening in the office next to you. There is an active Twitter scene of iGem teams that is really useful... with a knockout and expression construct on the same piece of DNA. We do a lot of protein-protein interaction...

...for cellular activation. In addition, FACS achieves greater purity of your sorted cell sample than MACS....selecting your cells of interest is magnetic bead-activated cell sorting, or MACS. But MACS usually only allows...a single (lineage) marker, while fluorescence-activated cell sorting (FACS) enables you to include further...indirect labeling is through antibody host species reactivity. Just like for western blotting, a primary epitope-targeting... T cells (Figure 2). Even better, we can use the same fluorophore for the three lineage markers we have...gating labels all unwanted cell lineages with the same fluorophore to easily gate on the unlabeled cells...different while the permeabilization solution can be the same). Examples of such targets include transcription...

...much more challenging to design a hands-on interactive activity using elephants than it is to design one...or do you need a full exhibit? In a five minute activity at a street fair, or a semester-long after school... difficult to reach people who are not already actively interested in scicomm. Difficult to do in-depth... allow people to learn in multiple ways from the same experience.  Can combines cons of multiple medias...

...exposed ssDNA bases. Thus, base editors have an activity window restricted to the stretch of ssDNA created... edits at certain positions within the editing activity window (Kurt et al., 2021). They hypothesized ...2022). Specifically, they found that the SOX2 activation domain (SAD), which recruits histone acetyltransferase...suggest that DSBs are a common intermediate of CGBE activity, and they are sometimes processed by error-prone... around the same time. (The papers were actually published in the same journal on the same day.) This ... But the same strategy in E. coli produced different results. GBEs seemed to follow the same initial mechanism...repair pathways within the cell — in some cases, the same repair pathways that were considered problematic...

...enzymes such as ClaI or XbaI, or, conversely, activating enzymes such as DpnI. For example, cleavage by...affected Not affected DpnI Requires methylation for activity Not affected Not affected If your enzyme is... experiment? The answer to both questions is the same--methylation! Read on to learn about how DNA methylation...companion endonucleases that recognizes and cleave the same DNA sequence if not methylated (if it comes from...Although not all prokaryotic DNA is methylated to the same level, the potential for methylation should be considered...any) of those sites will be methylated. Using the same example plasmid as above (which has the XbaI site...

...be developed and optimized for the samples, antibodies, activation, and assay readout used. You’ll note...antibody. Perform a second series of wash steps. Activate the signaling molecule and perform the assay readout...approach is as follows: Prepare the sample. Incubate the sample with the primary (unconjugated) antibody...if all the primary antibodies are the same isotype from the same species, only one secondary antibody ...remove any unbound or excess antibody. Incubate the sample again with a conjugated secondary antibody, which...primary antibodies indiscriminately, giving them the same output signal. In these cases, it is best to use...not allow for absolute quantitative analysis of a sample. Instead, relative quantification can be done using...

...Isaacman-Beck et al., 2019. Sparse Predictive Activity through Recombinase Competition (SPARC), a genetic...system to use other experimental transcription activation systems, such as LexA/p65 or mCD8/GFP, to allow...allows for the labeling of a subset of cells with the same genetic makeup, overcomes all of these labor-intensive...upstream of the second attP site to cleave and inactivate any read-through transcripts of the effector ...

... – placing Cre downstream of promoters that are active only in certain cell or tissue types, during certain... used for a wide range of purposes, including activating an oncogene only in a particular organ, or bypassing...two loxP sites, which can be located either on the same or on separate pieces of DNA. Both 13bp repeat sequences... sites: Inversion: If the loxP sites are on the same DNA strand and are in opposite orientations, recombination... reversed.   Deletion: If the sites face in the same direction, the sequence between the loxP sites is...sites undergo recombination with other sites of the same type, but are not cross-compatible. Using different...

...changes in autophagic activity, and are a more reliable indicator of autophagic activity. The aim of this ...mRFP-GFP-LC3 functions as an autophagy bionsensor via the same mechanism as Rosella: GFP is quenched in acidic ...

...substantial size of GFP may interfere with the activity or the localization of the fusion protein. Also... visualization of heterotrimeric G protein Gq activation in living cells." BMC biology 9.1 (2011): 32....al. "A FRET-based biosensor for measuring Gα13 activation in single cells." PloS one 13.3 (2018): e0193705... proteins are also a better choice for thicker samples. Another solution to avoid autofluorescence is ...reaction and hence, there is no need to excite the sample. Although bioluminescent probes are usually dim...

...of the optogenetic regulator leads to spurious activation of the FRET sensor. Researchers have sought to...Here the bioluminescent donor is unaffected by activation of other fluorescent probes but can still transfer...as the excitation wavelengths needed would also activate the optogenetic accuators. The researchers took...NLuc requires 100-fold less protein to produce the same amount of light from an Fluc or RLuc reaction. In...protein that could be co-imaged with GFP using the same wavelength for excitation. The result was the cyan-excitable... K., et al. (5) imaging multiple proteins at the same time in live cells using fluorescent proteins (FPs...and colleagues (8) were interested in using photoactivatable, optogenetic accuators to depolarize or hyperpolarize...

... This repeat region associates with the trans-activating crRNA (tracrRNA) which interacts with the Cas9...Modifications of CRISPR RNAs to improve Gene-Editing Activity and Specificity. JACS 144, 12584-12594 (2022).... 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...Another stabilizing sugar modification, found at the same site, is 2’-fluoro (2’-F), which substitutes a fluorine...protein and can increase on-target efficiency. In the same vein, locked nucleic acids or bridged nucleic acids...modification have overcome these woes with photoactivatable and photocleavable gRNAs. Photocleavable guides...

...variation in color depends on how transcriptionally active the genomic locus of integration is. The white ...TH17 cell differentiation by antagonizing RORγt activity. Nature. 472, 486–90 (2011). PubMed PMID: 21441909...recombination so that the transgenes can be inherited on the same chromosome. Targeted Insertion allows for direct...variants of YFG because the transgenes are found in the same locus across fly lines (i.e. there won’t be positioning...

...level, the Gal4/UAS system is a transcription activation system co-opted from yeast (1). It is a Drosophila...turn genes on or off. Gal4 is a transcriptional activator that binds to UAS enhancer sequences found in ...this mutant fly to one lacking the mutation in the same genetic background. In other cases, YFG or one of...

...hybridomas, immortalized cells created by fusing an activated B cell that produces the antibody of interest ...heavy and light chain, each from a constitutively active CMV promoter. Constructs were then verified by ...detect multiple targets with different mAbs in the same experiment. The Trimmer Lab found that they could...