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... to a method of physically joining multiple fragments of DNA to create a synthetically designed DNA sequence...-guide RNAs – but it also allows you to skip purification steps and end-repair steps with well-designed...fragment sequences and the polymerase used in the amplification. This tool allows instant updates as fragments...transforma­­­tion or as a PCR template. Some of the applications of the NEBuilder HiFi DNA Assembly Mix include...one single-stranded oligo, instead of the more typical two oligos, is more cost effective and eliminates...the limitations associated with otherwise more complicated assembly approaches, and opens the door for new...

...Musicophilia includes case studies of relevant neurological atypicalities, including a patient with amusia - the...capture the attention of even the most skeptical reader of historical fiction (I can personally attest to...sometimes we don't."     - Jennifer Tsang, Science Communications and Marketing Coordinator Non-science favorites...Strange & Mr. Norrell by Susanna Clarke "A quasi-historical/fantasy fiction. Two dueling magicians in the...times."     - Aliyah Weinstein, Marketing and Communications Manager Addgene's book club Indeed, reading...

... fused base editors (nCas9-BE), as Cas9 is mechanistically more favored for efficient base editing. They...been used for multiplex Cas9-nuclease editing. Typically, a single array encoding the tandemly assembled...released by endogenous tRNA processing machineries, specifically, RNaseZ and RNaseP. Although the Gao Lab demonstrated...packaged into viral vectors for potential in vivo applications or multiplex CRISPR screening.   Using tRNAs...multiplex genome engineering of crops or allow for sophisticated CRISPR screenings that would perturb the unprecedented...and efficient in plasmid and viral vectors. In applications when repetitive tRNA sequences must be avoided...construct DAP array for multiplex gene editing applications, we provide detailed manual and online design...

... necessary for many of these applications have lagged. Fixation, chemical staining, and light exposure...antibodies may just not perform as well in one application as another, so it is always best to have options...for mEGFP-HRas (from Plasmid 18662). See the Applications section on the Anti-GFP [N86/8R] page for more...30667360; PMCID: PMC6377228. Hyperfolder YFP- a chemically stable tool for microscopy by: Susanna Stroik...well-suited for the demands of advanced microscopy applications such as super resolution, electron, and expansion...Find YFP plasmids here!   Campbell, B.C., et al. Chemically stable fluorescent proteins for advanced microscopy...

...around these issues, but this solution wasn’t practical due to the low intensity of luminescent proteins...resulting in a fluorescent signal. Since their first publication in 2012, the Nagai laboratory has assembled a...multicolored nano-lanterns for use in various applications, including optogenetics, biosensors, and fusion...sensitivity and faster imaging compared to previous publications. They also developed calcium, cAMP, and ATP ...furimazine. In addition to the previously described applications, the enhanced signal of eNL allows their use...characterizing Nano-lanterns and eNL for many different applications. For long-term imaging, especially where photobleaching...not alter cellular physiology in your system. Optical sectioning cannot be conducted with luminescence...

...you're trapped in a pattern of small errors that significantly impacts your experiments. What do you do when...variables - your approach, your workflow, or your physical setup - to prevent the mistake, just like you ...differently.  Tools to Try  Checklists Found in surgical suites, air fields, and military bases, checklists...light-sensitive reagents” or “re-sterilize now.” Have a physical copy of the protocol when you work and make a ...time to do them carefully and thoroughly. Have a physical copy on hand as you run your experiments. Try ...your plate maps onto your plates, alongside your physical copy. If you’ve got messy handwriting, reach for...accuracy.  Bench setup Small adjustments in the physical organization of your workspace and your workflow...

...made of chemicals, which have an unfortunate tendency to react with other chemicals and biological structures...antibody out on the bench overnight. Do not panic! Typically, antibodies are okay if they’re left out overnight...then I would suggest either testing the antibody efficacy through a standard curve, another quick experiment... fluorophores, which work not by reacting to a chemical, but by emitting a specific wavelength of light...compensation.    Working with antibodies can get a bit complicated, but hopefully this helps you successfully select...

...appreciable levels of modification. Whole-genome sequencing of cells modified by CRISPR indicates that the consequences..."font_set":"DEFAULT","size_unit":"px","font":"helvetica","fallback":null,"variant":null,"size":18},"button_color...20nt-NGG] and [5’-CCN-20nt], as it is equally efficacious to target the coding or non-coding strand of ...desired location of edit; efficiency decreases dramatically when the cut site is >30nt from the proximal... via CRISPR cutting. For CRISPRa, it is most-efficacious to target a ~100nt window upstream of the transcription...sequence may not be available. Predicting gRNA efficacy We and others have examined the ability to use...needs to test in the lab. Importantly, for any modification of interest, it would be unwise to make conclusions...

... The previous post in this series described a practical approach to selecting a bright fluorescent protein... localized fusion proteins and do not require dedicated plasmids or constructs. It should be noted that...reached at high power may not translate to real applications in which far less power is used. For example...insight into the photostability in the intended application (Goedhart et al, 2012). The third issue relates...Selecting a photostable protein The issues with quantification of photobleaching rates can be summarized as...Amsterdam). He develops, characterizes and uses genetically encoded fluorescent probes. You can follow him...(University of Amsterdam) where she develops genetically encoded FRET based biosensors.     Additional...

... out large regions of DNA and has anti-viral application potential as a result. Fast facts – PAM requirement...once it is engaged by its target, it will non-specifically cut ssDNA. This feature is shared by other Cas...enzymes outlined below as well. Cas12a also has significant therapeutic promise due to the fact that it is...target sequence Similar to Cas12, it is also significantly smaller in size than traditional Cas9. Another... potential as a viral therapeutic that could specifically degrade target viral RNA. For more information... has been proposed to be useful for the same applications (RNA knockdown, RNA editing, etc.). The main... not display collateral RNA cleavage and is significantly less toxic to mammalian cells than Cas13 (likely...

...by guest blogger Alan Wong. The complexity of biological systems can hinder our attempts to study and ...combinatorial genetic networks underlying your favorite biological phenotype in a scalable way. This blog post will... genetics that underlie complex regulation in biological systems. The CombiGEM platform provides a means...sequences via oligo synthesis using the format indicated below and pool-clone them into the pAWp28 storage...realization of a variety of perturbations and applications in functional genomics, cell reprogramming, ...currently an Assistant Professor at the School of Biomedical Sciences of the University of Hong Kong. He is...technologies to interrogate and understand complex biological systems. He can be contacted at [email protected]. ...

...As evidenced by all the CRISPR publications, press, and plasmids out there, it’s obvious that CRISPR ... Not only is CRISPR having effects on various biological disciplines, the base technology itself is constantly... the Broad Institute, the Joung lab at Harvard Medical School, and the Doudna Lab at Berkeley set out ...afforded by gRNA binding at non-target sites would theoretically decrease off-target cutting. Figure 1...PubMed PMID: 26628643. Find plasmids from this publication at Addgene. 2. Nishimasu, Hiroshi, et al. "Crystal...PubMed PMID: 26735016. Find plasmids from this publication at Addgene. 5. Chen, JS, et al. "Enhanced proofreading...PubMed PMID: 28931002. Find plasmids from this publication at Addgene. Resources on the Addgene Blog Read...

...spacer "NNNTANNN", where "N" indicates which bases may vary from the canonical sequence. Among these are ...important plasmid elements – promoters, origins of replication, protein tags, and antibiotic resistance markers...creating (excuse the pun) specific, targeted DNA modifications in transgenic animals, embryonic stem cells,...proteins or co-factors, thus allowing for broad applications in a variety of experiments. LoxP (locus of ...sequence gives the loxP site directionality, and the canonical loxP sequence is ATAACTTCGTATA-GCATACAT-TATACGAAGTTAT... the loxP methods described above, a genetic modification can be restricted based on experimental constraints...

...are time-consuming, have been typically optimised for immortal/genetically-corrupt cell lines, lack scalability...alone in the cellular milieu. It is, therefore, critical to understand how proteins interact with one another... another (or with DNA) to perform diverse biochemical tasks in the cell. One of the most popular approaches... to tag the protein of interest with a small biochemical “epitope tag” such that these tagged proteins... purchased from various other suppliers with a typical turnaround time of 5-7 days. The crRNA and tracrRNA... to tag >30 genes/plate in the first shot with typical knock-in efficiencies of 10-20%. Together, our ... the non-HDR allele. For N-terminal tagging, specifically searching for error-free knock-in at both alleles...

...ago. I just recently started working in the pharmaceuticals industry after earning my Master’s in 2016 ...accomplishments and demonstrate your science communication skills. Know your audience. Connect your prior...presentation as an opportunity to demonstrate technical and transferable skills that make you qualified...perfect science talk Be prepared for potential technical difficulties. Have your presentation on a flash...codes for interviews. You can also ask HR for clarification on the dress code for the interview. Greet each... quiet place to take your video call.  Expect technical difficulties. To try and circumvent this, test...from HR carefully. And even if you don’t have technical challenges, your interviewers might.  You’ll probably...

...named according to the number of strands in the canonical beta-barrel structure. Most frequently, the proteins...pairs with N173 derived from Cerulean or Venus Identification of new fluorescent protein fragments for bimolecular...fluorescence complementation analysis under physiological conditions. Shyu et al. Biotechniques. 2006 ...fluorescent proteins for protein labeling and signal amplification. Zhou et al. PLoS One. 2020 Bo Huang Yellow...Yellow Venus pBiFC-VN173, pBiFC-VC155 Identification of new fluorescent protein fragments for bimolecular...split-FP options. While the BiFC interaction is typically irreversible and requires some time for chromophore...

...methyltransferase DNMT3A allows researchers to easily add biochemical marks to genetic regions from which where they...approach (spoiler alert: it’s dCas9… with no modifications). Ready to get started?   Figure 1:...eventually, develop treatments, we need tools to specifically edit methylation sites.  Step aside, transcriptionally... DNA. When dCas9 is bound to a target CpG, it physically blocks DNMT1 from that site. So, when DNMT1 methylates... methylates the genome during DNA replication, it doesn’t methylate sites blocked by dCas9. Within a few...Szyf 2021.   Let me leave you with a few practical considerations: Design your gRNA to overlap the...for dCas9 to interfere with DNMT1 even through replication. The dCas9 also needs to be removeable, otherwise...

... from E.coli, named TadA7.10 in ABE7.10)  a catalytically impaired Cas protein (dCas or Cas nickase)  ...accidental insertions and deletions (indel) by the catalytically impaired D10A nickase from ABE7.10 can be a ...the authors created ABE8 constructs using a catalytically “dead” S. pyogenes Cas9 (dC9-ABE8.x-m/d). The...even in adult patients. In order to analyze the clinical use of ABE8 for primary human cell editing, the... reduced off-target editing frequencies. For applications that require high DNA editing specificity, for...deamination, an important attribute for therapeutic application. ABE8s demonstrate an overall improved base editing...editors with increased activity and therapeutic application. Nature Biotechnology. https://doi.org/10.1038...

... Roska’s lab at the Institute of Molecular and Clinical Ophthalmology Basel recently took a different ...Strategy #2 (ProB): Create promoters using phylogenetically conserved sequences that are upstream of the...expression. Surprisingly, <1% of synthetic promoters replicated the expression specificity of the wild-type promoters...reproducibly labeled retinal cells can be found here.  Applications for synthetic promoter AAVs Besides restricting...synthetic promoters have a few other potential applications: Using promoter combinations for targeted expression... promoters expressed in. The lab tested this application using one promoter to drive expression of Cre-mCherry... rates of labeling are ideal of gene therapy applications, promoters that produce sparse labeling would...

...process involves a significant amount of QC. First, we perform Picogreen quantification on each and every... preparation process and includes Picogreen quantification of various intermediate steps, a sizing electrophoretic...electrophoretic gel, and qPCR quantification of the final libraries. A broader view of the whole plasmid...often find a few mismatches in the origin of replication or other common backbone elements. Since we’ve...annotated Genbank files that are useful for more complicated plasmids, like those that contain synthesized...sequence or plasmids containing genes with many modifications. We look for point mutations, truncations, and...