...so far! Why do we need PAM flexibility? IDT estimates that there is an NGG PAM every 42 bases in the...might be undesirable due to potential off-target sites or low predicted on-target efficiency. In cases ...aureus and N. meningitidis require different PAM sites than SpCas9, in this case, NNGRRT and NNNNGATT, ...activity on non-NGG PAMs, generating xCas9 3.0-3.13. To test xCas9 3.0-3.13, Hu et al. first restored catalytic... that the clones were capable of cleavage, they tested the clones against an NNN PAM library in bacteria...editing rate as SpCas9 at an NGG PAM. When they tested a variety of NG, GAT, GAA, and CAA PAMs, they saw...outperformed SpCas9-VPR in activating transcription. Sites containing NG, GAA, or GAT PAMs averaged 56-91% ...

...be activated from low fluorescent states to high fluorescence states, they can change from one fluorescent... light. The fact that blue light both activates and inactivates Dronpa is not ideal, since this means ...488nm blue light. The T203H mutation in PA-GFP generates a chromophore population that is more neutral,...but then further exposure to blue light also inactivates the chromophore, leading to a non-fluorescent...photoactivation. A high contrast ratio usually indicates that a PA-FP has a low level of spontaneous fluorescence...and display bright and photostable green and red states. Beyond these imaging applications PA-FPs have ... the National Academy of Sciences of the United States of America 101.45 (2004): 15905-15910. PubMed PMID...

...create notes directly from webpage clippings, photographs, and file attachments. Organize your notes into...was in one of those four white binders. Thirty minutes later you’re frustrated and no closer to starting...pages in your notebooks and go wild with sticky notes and labels. Color code your Post-its and write brief... brief descriptions on each. Place the notes sticking up out of the top of the notebooks, so that you ...notebooks, create checklists that link to other notes, and use the search bar to quickly find what you...the protocols, guides, or references that your labmates ask to borrow most frequently. Copy these into...the revision history. Now you’ll be able to make updates to protocols in real time. You may also want to...

...low-affinity sites, the proteins work cooperatively to separate nearby AT-rich regions of DNA. In a test tube,...Then, the ATP-bound DnaA binds multiple specific sites within the ori called DnaA boxes. These boxes come...controlled plasmids. Binding of IHF (green) redistributes DnaA (red) from binding primarily high-affinity...helicases to continue unwinding the strands. This creates two replication forks that move in opposite directions... to regulate DNA replication. DnaA negatively regulates its own expression, so high levels of DnaA act...methylating newly synthesized DNA, blocking binding sites, or trading out ADP for ATP to re-activate DnaA....binding and contributing to DNA unwinding. This promotes initiation at the plasmid ori, after which replication...

...enzyme sites. You can easily move (subclone) any piece of DNA that already has restriction sites on either...systems is that like restriction enzyme sites, type IIS sites can be found throughout DNA sequences. Thus...double stranded DNA (dsDNA) at specific sequence sites in the DNA. In restriction cloning, scientists utilize...either side of it into any plasmid that has the same sites in the same orientation within its multiple cloning...short length, it is also easy to add restriction sites to any piece of DNA during PCR amplification, allowing...important to note that restriction enzyme target sites can be repeated throughout a specific DNA sequence...already be surrounded by specific recombination sites (in this regard, not so dissimilar from restriction...

...recombination sites. These sites are target sequences of PhiC31. In the SPARC system, attP sites are located...-UAS system along with precisely truncated attP sites to control recombination of the gene construct. ...was developed by Thomas Clandinin’s lab, and incorporates several common genetic tools in Drosophila (...labeled. PhiC31, a recombinase. It specifically integrates an attP site into an attB site. In the SPARC ...irreversibly recombines one of the two competing attP sites to drive expression of either a stop cassette or...used SPARC to image the calcium response in the dendrites of T5 neurons by placing the calcium indicator...

... AAV variant, rAAV2-retro, that migrates retrogradely at higher rates than any other AAV serotype (Tervo...neurons. Neurons are composed of a cell body, with dendrites receiving incoming information, and a projecting...at the terminals of axons form connections to dendrites of proximal neuronal cells. In the specific example...anterograde transport relies on kinesin. Different rates of speed for various forms of retrograde and anterograde...infection to the central nervous system where it replicates, spreads, causes neurotoxicity, and is lethal...et al., 2008). Herpes Simplex Virus (HSV) also migrates through neurons where it can replicate and by ...anterograde tracer and injected at several hundred sites across the entire mouse brain. Traced neurons expressed...

.... Polyclonal antibodies, which bind to multiple sites on an antigen, are cheap and can help amplify a ...reporter. Finally, each new conjugate will have to be tested to ensure it doesn’t impact the antibody’s binding...400 kDa MES for proteins <40 kDa 180 V, 30 minutes Good shelf life; runs at neutral pH (reduced ...400 kDa MOPS for proteins > 30 kDA 200 V, 30 minutes Good shelf life; runs at neutral pH (reduced ...your membrane. It requires anywhere from sixty minutes to overnight to run, and is recommended for very...proteins. A semi-dry transfer is faster — 3–30 minutes — and uses less buffer, but it is not as efficient... These proprietary systems are fast (often <10 minutes) and simple, utilizing individually packaged membrane...

...Addgene. What you might not know is that Addgene distributes curated collections of plasmids as kits with ...pExpression vectors) plasmids. The type IIS restriction sites used in these vectors are of low abundance (meaning...yeast, mammalian cells, insects, and even Xenopus oocytes. The expression vectors additionally contain various...protein tags (His, Avi, and GFP) as well as protease sites to facilitate protein purification. We hope this... Using a variety of GFP and RFP fusions as their test genes, the researchers at the BIOFAB showed that...peptides to facilitate protein folding and/or protease sites to remove the epitope tags. Different plasmids in...library of eGFP-tagged human ORF clones to allow testing and comparison of different cell-free expression...

...landing sites,” then the recombinase handles the installation of the donor sequence. The latest work evolves...epigenetic silencer Recombinant antibodies for marking astrocytes MagIC-Cryo-EM for sample enrichment and structure...PRC2 with RvLEAMshort (1:6 molar ratio) with 10 minutes of glutaraldehyde crosslinking: representative ...sequences containing attB/P recombinase landing sites on opposite DNA strands. When the prime editor releases...sequence with the attB/P landing site. A recombinase integrates a sequence from a donor plasmid containing a ...resulting in the integrated sequence between two attR/L sites. Image reused from Pandey et al. 2024 under CC-BY...autoinhibition and stimulating the methylation of nearby CpG sites on the target DNA, and thus inactivating the promoter...

...Design-Build-Test-Learn cycles. If you’re not familiar with this, it’s a method for building and testing new products...that process!   Figure 1: The Design-Build-Test-Learn cycle adapted for video making.   Cycle...rolled several long takes, and we cut it up in post.  Test: Overall, it worked. We pulled it off! Check out...Nonprofit Day, which was the perfect opportunity to test shooting over Zoom, everyone’s new favorite third-party...to some volunteers, who filmed with their phones. Test: I loved the end result, but it was clear this wasn...functions, including starting and stopping recording).  Test: It was unbelievable how well this worked! Interviewees...would remain masked, it freed us up to be creative.  Test: Our first official shoot went off with almost no...

...conventional cloning (to introduce or remove restriction sites), in mapping of regulatory elements (to mutate promoters...parent vector). Also shown are the “B” restriction sites, which flank the targeted region. Upon PCR amplification... restriction site through mutagenesis vastly facilitates the subsequent process of screening for succesfully...maintained. This overlap ensures that the PCR generates a nicked circle rather than a linear product (...Figure B, digest A). In contrast, if your mutation ablates a restriction site, digestion with the appropriate...portion of the plasmid with a primer pair that generates a linear product. The mutated product can then...the original plasmid using the unique restriction sites. Alternatively, if you want to make your protein...

... µL size serotype testing) pAAV-CAG-GFP 50465 AAV8, 9 (20 µL size serotype testing) AAV-hSyn-EGFP ...size serotype testing) pAAV-CAG-GFP 50465 AAV1, 2, 5, rg (20 µL size serotype testing) pAAV-hSyn-EGFP...Our new AAVs include: Control AAVs for serotype testing jGCaMP7c for calcium detection PSAM4 GlyR for chemogenetic...control AAV that scientists can use for serotype testing. These samples are available in various serotypes...encoding GFP. Check out our inventory of serotype testing AAV New control AAVs Plasmid Serotype Name ...are especially useful for studies in non-human primates, where fusion fluorescent proteins can affect ...

...with >3,500 unique isoforms transcribed from these sites. The Zhang lab generated a comprehensive library...TF to determine how its presence influences cell fates. The atlas of directed differentiation To understand...endeavor, a map of possible TF-induced cellular states was generated (Joung et al., 2023). Fig....single cell RNA-seq was performed to map TFs to cell fates. (Adapted from Joung et al.)    This atlas linked...and down-regulated pathways. Individual cellular fates were then matched with candidate TFs responsible...initiate disease onset, providing both a tool to test prophylactics on and a program that any researcher... generate unique cell types. Of the 24 TFs they tested, 22 expressed markers consistent with the cell ...

...of DNA, or templates, are spatially separated from each other. These attached DNA templates are then amplified...Alternative start codons include CUG in eukaryotes and GUG in prokaryotes. Common Epitope Tags Tag Amino Acid...DNA. Although they can be found in archaea and eukaryotes, they play the most significant biological role...fragment with a plasmid or vector DNA backbone generates a recombinant DNA molecule, which can be used ...of DNA which contains several restriction enzyme sites, enabling easy insertion of DNA by restriction enzymes...the promoter. As a general rule, the restriction sites in the MCS are unique and not located elsewhere ...relatively quickly, and exhibit exponential growth rates, plasmids can be replicated easily and efficiently...

... event that homology-based searches return no candidates. Our strategy: Express random fragments of the...library worth using if at least 40% of colonies tested showed correctly sized inserts following colony...for single colonies (this usually requires 10-20 plates) (see box below). This initial transformation should...colonies (150-200 per plate) across 20 fresh selective plates. Step 2: Express random phage DNA fragments in...Several protein induction conditions may need to be tested, as with any protein purification, but overnight...protein induction is done, peel the membranes off the plates and place each onto a lysis solution-soaked filter... strongly to nitrocellulose, over-incubation of plates can lead to high background that obscures the desired...

...differences between these approaches, and provide updates on how best to design gRNAs. You can also find ...using S. pyogenes Cas9 (SpCas9), potential target sites are both [5’-20nt-NGG] and [5’-CCN-20nt], as it ...strand of DNA. As a rule of thumb, we avoid target sites that code for amino acids near the N’ terminus of...annotated start codon. Likewise, we avoid target sites that code for amino acids close to the C’ terminus.... For a 1 kilobase gene, since potential target sites occur ~1 in every 8 nucleotides, restricting gRNAs...this can decrease the number of guides one needs to test in the lab. Importantly, for any modification of...has been established, and can be used to identify sites that are likely to give rise to an off-target effect...

... handle most of these intermediates, with each one acting on specific substrates and in distinct cell ...stranded, while ssRNA viruses can generate dsRNA intermediates as part of the viral replication process. dsDNA...transcription. In an uninfected cell, dsRNA rarely circulates, but upon viral infection, this nucleic acid ...acid can build up. The foreign dsRNA activates the innate immune response machinery of the host. This pathway...virus, you could introduce some of its dsRNA intermediates into a healthy individual to provoke an immune...molecules designed to degrade mRNA transcripts. RNAi activates the RNA-induced silencing complexes (RISCs), which...DNA template, inherently generating RNA:DNA intermediates in the process. These transcription-coupled ...

... conduct microinjection experiments in S phase zygotes at 18 hr post fertilization. They observed higher...co-injected CRISPR machinery with sperm into M phase oocytes, thus allowing repair to take place before DNA ...Figure 1: Homology-directed repair in targeted zygotes proceeds from the maternal allele rather than the...embryos proceeded to the blastocyst stage at similar rates to untreated controls. Ma et al. performed sequencing...sequencing of 23 potential off-target editing sites in the embryos, as well as whole exome sequencing of ...However, it is important to consider that already-tested CRISPR alternatives are applicable in many situations...surrounding CRISPR-based therapy is the level of testing needed to define whether off-target editing or ...

...STEM graduates and postdocs in academia, industry and other areas of employment? How many graduates and...related to the structure of the biomedical workforce. Quotes taken directly from participants (summarized in...per PI” “Lack of awareness about how the system operates and functions” “Complete lack of information on...of academic research (4). There are currently 17 sites around the USA undertaking BEST programs, including...regulatory, or clinical environment. It remains contested, however, whether a better solution would be to...grassroots organisations that represent postdocs and institutes that employ them, the diversity of trainee backgrounds... and the many different paths that biomedical graduates take in their future careers. To participate in...