...overhangs with the same three nucleotides in a row; (4) no more than two nucleotides in the same position; (...that high-fidelity reactions could be done using overhang sets that broke rules 3—5. Fidelity, it turns... design high-fidelity Golden Gate Assemblies and overhang sets: NEBridge Ligase Fidelity Viewer™; NEBridge...reactions. NEBridge Ligase Fidelity Viewer This tool allows a user to check fidelity of an overhang set by ...remarkably high 71% fidelity, while the 52-piece assembly of a lac operon dropped to 49% fidelity, with significant...be strictly followed in order to generate high-fidelity reactions. (Need a refresher? The five rules are... a single tube. Their assembly had a predicted fidelity of 71% (Pryor et al., 2020). And in 2022, using...

... requires that you provide two out of six forms of documentary evidence: Evidence of receipt of major...obtain permanent residence. The recently established case Matter of Dhanasar provides the three-prong ...field Evidence of original scientific or scholarly research contributions in the field Evidence of authorship... to convey general information only and not to provide legal advice or opinions. The contents of this ...advice on specific legal issues.  The permanent residence “green card” process is a necessary and often ...uncertainty associated with filing a permanent residence case with United States Citizenship and Immigration...national researchers who require visas, permanent residence or citizenship to continue their work in the US...

...defined peptides. Munro and Pelham’s first published peptide tagging experiment involved a peptide from ...with unrelated peptides that already had high quality antibodies available - aka peptide tagging.  Key ...bound to the mid-protein peptide, while three bound to the C-terminal peptide. One of these C-terminal... of these new antibodies and identified Myc1-9E10 and its target peptide as a suitable pair for protein...from the neuropeptide substance P. This protein had a highly specific antibody to the C-terminal five amino...could be quite small. However, substance P has an amide group added to its C-terminus post-translationally...critical for antibody recognition. As such, the peptide could only be added to the C-terminus of proteins...

...for protein-peptide locking, Mark Howarth’s lab has developed a new tool for peptide-peptide locking. The...One 2013 Oct 7;8(10):e76117. Irreversible peptide-peptide ligation using SpyLigase Building off their...that promotes the formation of an isopeptide bond between 2 peptide tags, SpyTag and KTag. The group demonstrated... demonstrated the use of the SpyLigase peptide-peptide interaction to link affibodies or antibodies against...scientists to use CRISPR-Cas technology to perform genome-wide knockout screens. These vectors and sgRNA libraries... to browse CRISPR plasmids, watch informational videos, download protocols, and more! Looking for background... CRISPR technology? See our improved CRISPR-Cas Guide. Wang et al., Science. 2014. Jan 3;343(6166):80...

...libraries exist, but none provide a means to continuously diversify all nucleotides within a user-defined ...10-6 mutations per nucleotide per generation vs. 10 x 10-10 mutations per nucleotide per generation of ...diversification of all nucleotides at a user-defined loci in bacteria. Its modular nature provides versatility in...O., et al. "CRISPR-guided DNA polymerases enable diversification of all nucleotides in a tunable window... changes to a DNA sequence can help scientists identify and/or facilitate the evolution of new phenotypes...method developed by the Dueber Lab at Berkeley, provides a new approach for generating novel genetic variants...users to achieve targeted diversification of all nucleotides within a defined region of a genome. Require...

...even single mismatches within a CRISPR guide can prevent the guide from binding to the genome or prevent...thetargeting sequences of guides across 8 CRISPR libraries. Samples are divided into those of African ancestry...approximately 20% more single nucleotide polymorphisms (SNPs) that map to guides in the Broad’s Genomic Perturbation...accounted for in the CRISPR guides they used, leading to mismatches between the guides and the lines’ genomes... CRISPR screens in DepMap to see if they could identify the ancestry of the cell lines (European, East...germline genetic variations. They then wanted to identify cancer-associated genetic dependencies associated...relationship between the genetic dependencies they identified and their associated ancestries. While there ...

...adapted "self-cleaving" 2A peptides into their muliticistronic vectors. These peptides, first discovered in ... these peptides are thought to function by making the ribosome skip the synthesis of a peptide bond at...cleavage with some of these peptides, no consensus has been reached on which peptide works best. Likely the...from a single transcript. The two strategies most widely incorporated into plasmids for research purposes...the specific genes cloned into the vector.3  2A Peptides To overcome some of the disadvantages of the IRES...between the end of the 2A sequence and the next peptide downstream.4 The "cleavage" occurs between the ...Proline.  The table below lists the four common 2A peptides employed by scientists. 2A cleavage is universal...

...menu. Typically, we use the default nucleotide database “Nucleotide collection (nr/nt)” as it contains ... the Basic Local Alignment Search Tool (BLAST) provided by NCBI. BLAST helps us compare the sequencing...reference sequences, such as full plasmid sequences provided by the laboratories that deposit their plasmids...programs available, we primarily use Standard Nucleotide BLAST (blastn), Standard Protein BLAST (blastp...BLAST (blastx). NCBI has a terrific getting-started guide for BLAST, which includes a simple explanation of...BLAST search pages. At Addgene, we use blastn to identify any discrepancies in Sanger sequences, such as...ORFs) and determine the potential effect of any nucleotide discrepancies. The blastp and blastx programs...

...A,T or C – but avoid NAG) or guide RNA target (“seed”) sequence. The idea was that these “blocking mutations...mutation), blocking the guide RNA binding site works well. However, since some guide RNAs may tolerate a ...end-joining (NHEJ) pathway, leading to nonspecific nucleotide insertions, dele­tions or other mutations, referred...repair template, such as a sin­gle-stranded oligodeoxynucleotide (ssODN), which contains the intended sequence...efficiency.   But what’s better: PAM-blocking or guide RNA blocking mutations? This depends entirely on...however we have also demonstrated the efficiency of guide RNA blocking mutations. So if the locus does not...while taking advantage of the improved efficiency provided by blocking mutations. Essentially, you:  Step...

...social media, videos, newsletters, guides, and e-books – comes into play. Our goal is to provide informative...using the example of our Antibody Guide, which was published alongside our new antibody services in the... The guide was then assigned to our science communication specialist, Rachel, to write. Identify expertise.... Since our guides are heavily used, our graphic designer made most of the Antibody Guide figures. This...topics for the Addgene blog? Or why we make certain videos? In this post, we invite you behind the scenes ...you, whether you realize it or not! Many of our ideas come from social media conversations, conferences... all of our frequently asked questions!    Identify needed content Let’s take a look at how we create...

... that you provided for each sample. Guide Target - This is the user-defined 17-23 nucleotide sequence ...sequencing files and provide basic information such as your sample names and guide sequences, and ICE will...which also includes our CRISPR knockout guide design tool, guide validation tool, and guaranteed editing... Lead at Synthego. Article source: Step-by-Step Guide for Analyzing CRISPR Editing Results with ICE on...editing field by making experimental workflows considerably easier, faster, and more efficient than previous...examined ICE analysis of Sanger sequencing data alongside the analysis of NGS-based amplicon sequencing ...time or as a batch of hundreds, and indicate the guide RNA sequence you used. The ICE tool will calculate...

... more fully identify with and be inspired by the mentor’s successful example. For evidence of this, you...Valencia): “IMP showed me a wide range of professional opportunities that are outside of our typical boundaries... Program (IMP) USA-Europe was created. IMP as an idea was conceived and developed by its Director, Dr.... closer than ever (although not physically) the ideas and experience from talented professionals to their...together with the universities, a bright future. IMP provides matched one-on-one international mentorship, with...globalized world where individuals can exchange ideas, network, collaborate, and innovate internationally...offered by programs like IMP USA-Europe not only provide the specific technical and logistic support to ...

... particular, one slide of the presentation made a lasting impression on me. The slide showed a complex...approach is to identify an appropriate list of genes to target. Thus, it is always a good idea to keep a bioinformatician...Sweden. It can be very time consuming to design 5 guide RNAs (gRNAs) targeting each of the 1000 genes you...screens targeting a (long or short) list of genes provided by the user. This approach is useful for several...effective by world class CRISPR labs.  Check Out This Video to See How Green Listed Works at a Glance!  CRISPR...subsequently sequenced by next generation sequencing to identify which gRNAs are differentially expressed in the...to dissect a complex signaling pathway, and to identify what part of a pathway to inhibit to block the...

...joint. Silly ideas (mining local salt deposits to sell at farmers markets), and serious ideas (how to stop...for the future. From good ideas to a SynBio startup At some point, our ideas got pretty good. We started...journal clubs outside of work. Our scientific training was put to use as we explored ideas, planned out...we could do to make ourselves marketable to the wider world and occasionally thinking about how we could...establishment hiring system altogether. We had a lot of ideas about things we could do for work, and they weren... together we make a pretty good team. We have confidence that we can take on any challenge and quickly...marketable product. During this process, we had an idea that survived the literature review, survived technical...

...chain-terminating or dideoxy sequencing, was a major breakthrough for determining the order of nucleotides in a string...Sanger sequencing uses modified nucleotides, called dideoxynucleotides (ddNTPs), lacking the 3’ hydroxyl...added and when. Compiling the identity and location of a given nucleotide, a researcher (or a simple computer...the widespread use of Sanger sequencing. Building upon this technology, radiolabelled nucleotides were...Edward M. Southern (1), can be used to detect DNA identity, size, and abundance. If a scientist wants to ...gene mutation, southern blotting can be used to identify the presence of that mutated gene. In southern...One example of southern blotting being used to identify a gene mutation is the genetic test used for sickle...

...whenever applicable?Against this, consider the cost of an accidental release. Science relies on popular... DNA cassette encoding the cas9 gene and single guide RNAs (sgRNAs) with appropriate flanking homology...homology into a germline cell can create an RNA-guided gene drive. In principle, this event could happen even...available. Consequently, the best way to avoid accidentally creating a gene drive is also the easiest: don't...barrier protocols, they can reduce the risk of accidental escape to a negligible level. One major reason...publish the concept and likely capabilities of RNA-guided gene drives in the summer of 2014 (after extensive...hope was that anyone else who came up with the same idea or wanted to build a gene drive in a model organism...

...to the guide RNA, Cas9 cannot cut. 2. Bind to target DNA in the presence of a guide RNA provided that target...the components necessary to: 1. Bind to a guide RNA The guide RNA, which will be described in more detail...of the site targeted by the guide RNA. In the absence of either the guide RNA or a PAM sequence, Cas9 ...synthetic guide RNA or gRNA (sometimes sgRNA) In the native Type II CRISPR/Cas system, Cas9 is guided to its...into one RNA sequence known as the guide RNA (gRNA) or single guide RNA (sgRNA). Throughout the remainder...Cpf1) to cut the genome and a small RNA (gRNA) to guide this nuclease to a user-defined cut site. After ...Please note that while this post is intended to provide a general overview of CRISPR components, new Cas9...

...Producing stop motion videos is very time consuming; producing a 1-2 minute video takes an entire week ...with the images thereby making the videos more real. To give you an idea of the amount of work, see how many...’s gotten easier now that I identify with the main character in the video. We’ve grown attached to the...Dalila Cunha de Oliveira. Bricking Science is an idea built, literally, 'brick-by-brick' to introduce ...mysterious source of failure the lab gnome. See this video where he/she/it/something is captured on camera:...most people don’t get to experience this very human side to scientific research. Find Protocols Lab Tips ...was born. We chose the stop motion format for our videos as it is the most approachable for LEGOs. Stop ...

... development. Paula Videira: Congenital disorders of glycosylation Paula Videira is an Assistant Professor...been working with the Rare Genomics Institute to provide plasmid grants to researchers working on rare diseases...through the BeHEARD Award. Congratulations to Paula Videira, Michael McMurray, and Richard Gronostajski who...steps in the glycan biosynthetic pathway and have a wide range of clinical manifestations. PMM2-CDG is the...) activity due to mutations in the PMM2 gene. Videira’s group recently reviewed and reported that PMM2...studied and understood.  In line with this, Prof. Videira and collaborators will use Addgene plasmids to ...altered in PMM2-CDG. These studies are expected to identify new therapeutic targets/solutions and enable scientists...

...the bulk of the peptides identified to be from the target protein. This approach is ideal for validating...the sample was identified as your target protein, allowing you to proceed with confidence.  Share your ...experiments that you’ve been planning, it is a good idea to pause and make sure that your antibody has been... appropriately validated so that you can have confidence in your results. But what is appropriately validated... This post will go over some of the general considerations you should review before using an antibody.... that your antibody is not binding to proteins outside of your target protein. Show suitability for an...There are a number of resources available that provide in-depth discussions on strategies for antibody...