... and a downstream constitutively active eGFP. To create the inactive mCherry, the lab introduced a 43 .... Cells can then be sorted using Fluorescence-activated cell sorting (FACS) and the editing efficiency...system to identify and enrich for base-editing activity The reporters mentioned above all rely on GFP ...reporter system to identify and enrich base editing activity. Nucleic Acids Research 48:2841–2852 . https:/...by canonical base excision repair enzymes. At the same time Cas9n cleaves the opposing DNA strain which...to eliminate fluorescence. This created three inactivated eGFP reporters- eGFP L202, eGFP L138, and eGFP...

...Amplification Protocol (DOCX, 15 KB) CRISPR Activation Human Genome-wide CRISPRa-v2 Libraries 104,540...Re-Amplification Protocol (PDF, 71 KB) CRISPR Activation Human Subpooled CRISPRa-v2 Libraries 12,285...Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening, a protocol paper by the Zhang lab,...library from Addgene, they’ll need to amplify the sample for further downstream applications. As individual... all of the bacteria can grow and compete in the same container, making it easier for a smaller, recombined...

...within the target gene. CRISPR can also be used to activate or repress target genes without permanently modifying...CRISPR libraries have been created to knock out, activate or repress target genes by combining a gRNA library...Addgene carries CRISPR libraries for gene knockout, activation, repression, and barcoding.  3) Are you trying...Enrichment or depletion of multiple gRNAs targeting the same gene can be strong evidence that a particular gene...

...deposited PSAM AAV, which encode engineered ligand-gated ion channels that can be activated with FDA-approved...expanded our inventory of chemogenetic AAV for activating and inhibiting neurons. Minimally-tagged DREADD...size 20 µL samples of control AAV that scientists can use for serotype testing. These samples are available...serotype testing jGCaMP7c for calcium detection PSAM4 GlyR for chemogenetic inhibition Flp expression ...ability to detect the neurotransmitters and other bioactive molecules is integral to the study of neuroscience...Plasmid Serotype Name 119741  AAV5  AAV SYN flex PSAM4 GlyR IRES EGFP Recombinase AAV Finally, we are...ppAAV-CKIIa-stGtACR1-FusionRed New tetracycline transactivators Plasmid Serotype Name 99120  AAV1 pAAV-ihSyn1...

...original method, we used the Tetracycline trans-activator system to boost the expression levels, and we ...we named the system Tetbow (Tetracycline trans-activator Brainbow) (Sakaguchi et al., 2018) (Figure 1)....not powerful enough for large brain samples. For large brain samples, we recommend pretreatment with ScaleCUBIC1...time points, sacrifice the animals and fix the samples. When you use AAVs, it typically takes a few weeks...excessive amount of XFPs expressed. Then, clear the samples and visualize. For more details on this procedure..."Optical clearing and index matching of tissue samples for High-resolution fluorescence imaging using ...

...Similarly 90% of active research scientists believe climate change is mostly due to human activity while much...opinion do not seem to take note. For example, 91% of active research scientists (scientists who hold a doctorate...conversation about science’s role in government as well as active advocacy will be required. Information on how ...politician Rush Holt said, government must have the same fundamental attribute that enables science: “humility...

... scared to engage with them. Comment on their activities and participate in discussions. Get your voice...Twitter to promote your other science communication activities, e.g. tweet it when you publish a new blog post...step I have taken in my career. The moment, I got active with it, new doors started opening up. My current...requires lots of data. Get your community involved in sampling or bioinformatics analysis or upload your data...

...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 ...

...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 ...

... 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...

...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...

...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...

...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...

...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...

...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...

... – 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...

...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 ...

...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 ...