...non-coding RNAs (lncRNA). lncRNAs are over 200 nucleotides in length and lack an open reading frame, so ...transcription machinery of a target gene or DNA that codes for ncRNA. CRISPRa, on the other hand, recruits ...which targets RNA instead of DNA and selectively degrades RNA molecules. This system is often used to knock...

...throughout the library preparation process and includes Picogreen quantification of various intermediate...sequences for Illumina sequencing as well as unique barcodes per sample so we can assign sequencing reads to... use a modified version of the Python program described by Joung et al. 2017, which we hope to make available...

...plasmids and pooled libraries were originally described in a peer-reviewed scientific publication. We ...scientists answering their technical support questions (besides Addgene, I know both Abcam and NEB have Ph.D.s ...believe that scientists who contact customer service deserve prompt, courteous responses. No one is likely to...

... try amplifying the viral vector with bacteria designed to minimize rearrangements such as NEB Stable ...production of virus. Alternatively, if the virus encodes a fluorescent protein, the packaging cells could...working with, there’s a lot that goes into virus design, production, and transduction. Once you’ve harvested...

...positive and negative populations, Careful panel design In both conventional and spectral flow cytometry...indistinguishable. This tool is of particular value when designing a panel.     Figure 3: Similarity index...if markers are co-expressed in your cells when designing your panel. If you have two highly overlapping...

... This vastly increases the possibilities when designing screens. Each serotype has its own tropism, or...experiment. Additional considerations One of the downsides to using AAV is the limited packaging capacity...Adames, A. C., Saur, D., & Rad, R. (2022). Tutorial: design and execution of CRISPR in vivo screens. Nature...

...essence, pluripotent cells can be thought of as descendants of totipotent cells. An example of pluripotent... produced Ethical concern High. Embryos are destroyed during ESC isolation. Low Chances of post-transplantation...R., M.W. Beckmann, and W. Wurfel, Non-embryo-destructive Extraction of Pluripotent Embryonic Stem Cells...

... also the easiest: don't use a DNA vector that encodes both Cas9 and sgRNA. What if I want to work with...one mistake can be enough. Reversal drives are designed to overwrite a previous gene drive and thereby... on popular support, which in turn depends on widespread trust in scientists to develop new technologies...

...terrific getting-started guide for BLAST, which includes a simple explanation of the different BLAST programs...matrix BLOSUM62, but you may want to check the description of the other matrices to see if another would...alternatives Depending on your sequencing result and desired analysis, BLAST may not always be your optimal ...

... can teach  5-10 year olds in a single evening. Grades K-5 cover a broad range of attention spans, skill...CRISPR work? This section was essentially background slides getting everyone on the same page in terms of the... by running it by the teachers and sharing some slides on Twitter, where I was able to get some great ...

...their production requires animal sacrifice and precludes genetic engineering of a tag or fusion with a ... with secondary nanobodies conjugated with the desired fluorophores. In figure 4B below, triple colocalization...review. If you just want to use the nanobodies described in Pleiner et al, the key steps for generating...

... the data points generated by replicate values provides useful information about the accuracy of the curve...If the unknown sample’s measurement is too low, redesign the standard curve dilution series with lower ...often recommend using advanced graphing software designed to plot complex trendlines. If you don’t have ...

...The NPTII gene confers resistance to several aminoglycosides, including G418 which is commonly used for ...DNA and generating DNA breaks. The Sh ble gene provides zeocin resistance by expressing a protein that...experiments in which off-target mutations are undesirable. Pros: Works across many species/systems. Cons...

...Microscopy. What is Expansion Microscopy?  ExM provides a straightforward answer to the space problem....improve label density (Mikhaylova, et al. 2015).  Despite these limitations, ExM is a valuable tool, especially...Bournonville L, et al (2021) Expansion microscopy provides new insights into the cytoskeleton of malaria ...

...semester one and then proceed to use those skills to design and carry out their own novel experiments in semester...resources, offers mentoring for instructors, and provides opportunities for students to exchange ideas and...results in student learning gains and positive STEM attitudes in CUREs. CBE Life Sciences Education. 21(4):ar74...

...Origin and purpose of MXS chaining MXS-chaining was designed to create plasmids for fluorescence imaging or... can be assembled. For example, because CpG dinucleotides are underrepresented in most vertebrate genomes...and MluI recognition sites, which contain CpG dinucleotides in their 6-mer sequences, are rarely found in...

... Protein tags are usually smallish peptides incorporated into a translated protein. As depicted in the...depending on their protein of interest. After several decades of trying to address these challenges, researchers...examples. The tables list more common tags than are described in the post, but have been categorized to help...

...worked extensively with enChIP systems. Can you describe this technology and its advantages? Fujii: In ...enChIP system using CRISPR technology. Can you describe this technology and some of its potential applications... any other technologies that you would like to describe?  Fujii: I believe that "Biology is Method", i.e...

...biochemical properties. In 1995, Roger Y. Tsien described an S65T point mutation that increased the fluorescence... in the wildtype protein and facilitating its widespread use in research. Many other mutations have since...array of GFP-based fluorescent biosensors has been designed to detect a variety of intracellular conditions...

...Usage Tabulated from GenBank database (CUTG), that provides an electronic dataset for codon-usage of 257,468...(A) The codon wheel depicts the 64 codons that encodes the 20 amino acids and three stop codons. (B) Example...Codon Optimization  ATUM DNA2.0  IDT Synthetic gene design made easy GenScript- GenSmart Codon Optimization...

...series of markers are set up sequentially, from broadest to most specific marker, and are represented as...are, try drawing a quadrant over the plot that divides it into four, using the location of the cell populations...Gating and plot orders  As the gates progress from broadest markers to most specific, the first step is to...

...most situations.  A slightly different method is desirable if the antibody is expected to bind weakly to ...vary in shape and structure. Their porous surface provides a large area for binding, but antibodies conjugated... slightly higher, than those of agarose beads, despite the size difference. Magnetic beads are particularly...

... If not, our protocol on Lentivirus Production provides a general approach for producing lentivirus in...excellent protocol paper by the Feng Zhang lab describing construction, lentiviral production, transduction...the Broad Institute- A variety of protocols and guides regarding pooled libraries, from cloning a pooled...

...to distinguish from background tissue unless destructive ultrasound imaging was used. We set out to develop...acoustic properties of these gas vesicles made non-destructive specific imaging in tissue difficult. The first-generation... allowed the GVs to be flexible and produce nondestructive nonlinear ultrasound contrast. It turned out...

...promising tool for future structure-guided drug design and studies of protein ubiquitination and degradation...CRBNmidi plasmid here!   Kroupova, A. et al. (2024). Design of a Cereblon construct for crystallographic and...specifically for C. elegans (Cao et al., 2024). The team designed coding sequences for mScarlet3 and mScarlet-I3...

...CRISPR-associated transposases” or MUCICAT. Here, we’ll describe each of these CRISPR systems. The original pCas...by sacB counterselection.  The MUCICAT toolkit includes versions of plasmids with different induction ...mechanisms behind their distinct phenotypes and to design a new generation of engineered cells for green ...

...other words, your optogenetic experiment should be designed around a behavioral test in which your circuit...of mouse transgenics. Everything that has been described is intended for the mouse; however, all the steps....Diester, Ilka, et al. "An optogenetic toolbox designed for primates." Nature neuroscience 14.3 (2011)...

...human cells (Kim et al, 2016). The Waldman lab has designed their AAV tagging vectors (pAAV-SEPT-Acceptor ...vectors for your specific gene, the lab recommends designing homology arms with the modification of interest...combining the AAV and CRISPR-based approaches is described in several recent reviews (Howes and Schofield...

..., it’s essential to understand them in order to design the most specific assays possible. In this blog...-target effects should impact your experimental design differently. Yes, it is always better to reduce...introducing a specified edit). However, if your design does have wiggle room, you can minimize off targets...

...facilitate the selection of cells containing your desired edit. The selection is marker is then seamlessly...sites is cytotoxic for proliferating cells and provides a simple tool for conditional cell ablation.,”...Zinc-finger nuclease-induced gene repair with oligodeoxynucleotides: wanted and unwanted target locus modifications...

...experiments to minimize it. Overview of DIO/FLEx vector design Recombinase-dependent vectors utilize pairs of ...between them. Early recombinase-dependent vector designs used a lox-STOP-lox (LSL) or frt-STOP-frt (FSF)...you intend to use performs in your system. This includes a thorough testing of both the serotype and promoter...

...good starting point for most researchers as it provides a list of putative off-target sites that they ... approach misses larger mismatches (e.g. six nucleotides) that may still lead to off-target double-stranded...contains both the rare off target event and the desired edit is low. Therefore, unbiased sequencing may...

...tetroxide). These conditions destroy most PA-FPs, but, in 2015, the Looger Lab designed 2 variants of EosFP that...to green). For example, mEosFP (named after the goddess of dawn in Greek mythology) switches from green...

...in the development of CBEs and ABEs. This post includes a lot of acronyms, so we’ve included a glossary... in different cell types, available at www.crisprbehive.design. CGBEs fused to pioneer factors to improve...strand that does not base pair with the guide RNA. Despite being called the "non-target strand" in CRISPR ...

...second edition includes new and updated content in a clean and easy-to-navigate design. You can find it...

...quest, which led us to Seqera Labs, a company that provides open source workflow orchestration software for...on how Addgene has used Nextflow to improve and redesign our pipeline. In it, they break down the unique...

...#12DaysOfCRIPSR hashtag to share new CRISPR content designed to help you with your CRISPR experiments and to...CRISPR, Addgene gave to me: tips and tricks for designing gRNAs for CRISPR editing. On the fourth day of...

...distribution. Lab tip #3: Barcode everything Use barcodes and scanners to keep track of reagents in your...materials quickly and easily. Learn more about using barcodes. Check out our “Lab Tip” filter on the blog...

...anniversary! We've done so much in the past two (2!) decades that it's hard to summarize, but we wanted to mark...experience we're sharing through blogs, eBooks, videos, guides, protocols, and of course, the friendly folks at...

...and find our guides, plasmid kits and other resources for microbial research. Kits, guides, plasmids, ...