...sites so that scientists can easily clone in their desired DNA sequences. Gateway technology relies on the...coli strains, most colonies should contain the desired, recombined construct. Read our recent Plasmids...where we might use Gateway cloning to generate our desired constructs: lentiviral expression of the human ...There are a few different ways to generate our desired entry clone - human KRAS flanked by attL sites....donor vector.   Method B: TOPO-cloning of the desired insert into an attL-entry-TOPO vector. TOPO cloning...depend on a number of factors, like your organism, desired expression level, and experimental purpose. For...and destination vector creates two products: the desires expression clone and a byproduct containing the...

...and not require interaction with the data authors. In some types of research, raw data requires huge amounts...Since early March, the open access publisher has required authors to include a note as to where readers ... formal statement that is already practiced or required by many other journals and several funding agencies...statistical methods, omission of some data, etc.  The firestorm that followed implementation of the policy comprised...be accessed. Adding to this burden, the policy requires that data should be available for anonymous request...evaluate the interpretation of the experiment would require access to each individual video. To PLOS’s credit...not finalized its definition of necessary data requirements and likely will not do so (calling it a “sisyphean...

...limited by the low editing efficiency of homology-directed repair (HDR), the pathway for resolving DNA double-strand...backbone, sidestepping the problem of HDR vs. NHEJ entirely. The first two classes of base editors were cytosine...changes. Solid red arrows indicate a conversion directly catalyzed by the base editor. Dotted red arrows...does base editing work? Base transition editing requires three elements. They are, broadly: A Cas protein...fourth-generation base editors, BE4, reduce the undesired C→G or C→A conversions that can happen with BE3s...editing efficiency (Koblan et al., 2018). Later directed evolution efforts yielded an editor with more ...et al., 2017). Creating an adenine base editor required an additional step because there are no known ...

...travel be required or will you stay in one location? How long is the commute? Will the job require public...Science careers divided by the most pertinent skills required Research skills The ability to think critically...and setbacks. There are many roles that actively require these skills, including: Science Reporter Scientific...accuracy and attention to detail are valuable skills acquired from a science degree that can be applied to many... Experience in the field or in a position that requires travel can show that you are self organised, reliable... Zoological Worker Conservationist Documentary Director Parasitology Botanist Nature Photographer/Artist...employers. Increasingly, computer skills are highly desired, as roles are becoming more reliant on technology...

...into proteins are steps in that direction (Chatterjee et al., 2013; Hilaire et al., 2017). GFP can only be...myristoylation requires an N-terminal consensus sequence (MG-) and prenylation requires a C-terminal consensus... 2002). GFP needs oxygen Chromophore formation requires a fully folded beta-barrel structure, followed...chromophore (Tsien, 1998). Importantly, this reaction requires molecular oxygen and as a consequence GFP remains...proteins.Therefore, GFP is not suitable for applications that require anaerobic conditions. In experiments requiring ...are generally added to the bathing solution and require uptake by the cells. Which of these strategies ... et al. (2016). To date, small probes that are entirely genetically encoded do not exist, but the development...

...government cannot declare that gravity change direction and make things fly up when we drop them. Scientific... their side of the bargain and do the research required to inform the public (8–13). Facing the possibility...consequences While frustration and anxiety have inspired people to rally for science, others are concerned... to take place around the world. There is no requirement that marchers or supporters be scientists – on...science is done by people like them. This, when paired with long-term science outreach, may have the most...treatments promised by modern research, more funding is required. This is by no means an exhaustive list and every...science rallies scientists to political action, and inspires politicians and citizens to include science in...

...targeted Cas9 cleavage, and repair through homology directed repair (HDR) and non-homologous end joining (NHEJ...created SRIRACCHA in 2017 to enrich and identify desired CRISPR mutants (Wen et al., 2018). SRIRACCHA uses...constructs for the reporter to work and to make the desired genome edit:  A donor plasmid that contains a ...OH-T and doxycycline. A gRNA that targets your desired gene (whose sequence is also found in the reporter...the reporter gene that was cleaved by Cas9 and repaired via HDR using the supplied donor plasmid will ...presumably the endogenous target site will be cut and repaired by NHEJ as well. The ratio of RFP to GFP expressing...integrated GFP reporter leaving you with just your desired genomic edit.  Find SRIRACCHA plasmids at Addgene...

... homology directed repair (HDR) to incorporate new information into DNA. This also requires an exogenous...lightly! Indeed, truly achieving the gold standard requires not one but two rounds of single cell cloning ... of target site is far more constrained by the desired location of edit; efficiency decreases dramatically...limited to single nucleotide transitions but still requires a nearby PAM, although these are still early days...to optimize numerous parameters to generate the desired edit. The wrench: Gene activation and inhibition...technologies – a nuclease-dead Cas9 (dCas9) is directed near the promoter of a target gene. Here, the ...the FANTOM database, which relies on CAGE-seq to directly capture the mRNA cap, provides the most accurate...

...correspondence via e-mail throughout the entire year. In addition to their direct training role as mentors to Spanish...Co-founder/director of the non-profit foundation IMFAHE and its International Mentoring Program Director of Strategic... as an idea was conceived and developed by its Director, Dr. Zafira Castano Corsino, a junior faculty ...’s Hospital (BWH). Based on her experience co-directing Mentor Circle Programs at BWH and providing career...supported, and can more fully identify with and be inspired by the mentor’s successful example. For evidence...mentors also facilitated the development of bidirectional USA-Europe professional collaborations, setting...their pre-program self-assesment. Achievements directly attributable to participation in the program included...

...enzymes, which have a flexible or non-existent PAM requirement, and how these proteins are advantageous in today...sequence is suitable, but there is no PAM next to the desired cut site, then a further-away gRNA where there ... may wonder: how close is close enough for the desired edit to still occur if the cut site is sub-optimal...it depends, but generally the efficiency of the desired edit is reduced by over 50% when the cut site is...there is a need to eliminate or reduce the PAM requirement to allow flexibility in gRNA selection and subsequent...constraints, or at least has looser or different requirements. Luckily, there are some available.   Cas proteins... generating Cas enzymes with alternative PAM requirements (Zhao et. al, 2023). xCas9 has been shown to...

... Park.  In addition to Taq DNA polymerase, PCR requires free nucleotides (dNTPs), template DNA to amplify...are crucial for this process as DNA polymerases require an existing strand of DNA to add nucleotides to...exponentially replicate the target DNA of interest. This entire reaction used to be done manually in water baths..., or for generating mutated sequences for site-directed mutagenesis.  Quantitative polymerase chain reaction...measuring a starting DNA concentration using PCR. qPCR requires the addition of a probe based fluorescent dye ...transcription-qPCR (RT-qPCR). The first step of this process requires RNA to be converted to cDNA using reverse transcription...rare alleles or mutations. ddPCR also does not require a reference or standard curve, which can be time...

...currently requires additional experimental manipulation. In some cases, injection of Cre is required, however...selection marker (Kim et al., 2014). Meanwhile Andy Fire’s lab used oligo-mediated knock-in of a dominant ...development of this approach reported in 2017 uses the direct injection of a CRISPR/Cas9 ribonucleoprotein complex...advantage of being used in any genetic background, but require variable amounts of experimental manipulation and...– to work with inefficient sgRNAs in my initial direct screening efforts, which were similar to the approach...products used to identify a single 2xFLAG knock-in by direct screening. The center images depict the power of... DNA double-strand breaks up to 54 bp from the desired insertion site result in efficient editing. Repair...

...-use of existing elements. DNA sequences for a desired component are cloned into Golden Gate entry vectors...expression to degradation. Chemical approaches may require expensive peptides or small molecules and exceed...on or off the expression of shRNA. This system requires less work than using established methods and minimizes...thorough understanding of complex biological systems requires both a catalog of molecular players and contextualized...fragments contain 482bp of overlapping sequence that direct Homologous Recombination (HR) or Single Strand ...strain EcAR7) and minimizing the set of plasmids required to make singly or multiply phosphorylated proteins...orientation, so that multiple DNA components can directionally assemble in a single reaction, while retaining...

... in Gene Therapy, Director of the Grousbeck Gene Therapy Center, Associate Director of the Ocular Genomics...Addgene welcomes three new members to the Board of Directors: Khandan Baradaran, Frank Stegmeier, and Luk Vandenberghe...of Quality at Dimension Therapeutics, which was acquired by Ultragenyx Pharmaceutical in 2017. As one of...In 11th grade she heard a talk about Z DNA that inspired her to major in molecular biology in college. ...funded,” she says. Baradaran met Addgene Executive Director Joanne Kamens several years ago through the Healthcare... an HIV lab and based on that experience, soon directed himself to finding “immunological and virological...commercialization efforts in gene therapy either through direct industry collaborations, grants, or setting up ...

...choice. This step is sometimes also referred to as “directed differentiation.” Specific cell media, supplements...transdifferentiation adult somatic cells are converted directly into a different cell type, bypassing the lengthy... carrying the transdifferentiation factors are directly injected into the organ of interest for in vivo...Highly efficient reprogramming to pluripotency and directed differentiation of human cells with synthetic ....N. Davis-Dusenbery, and K.C. Eggan, SnapShot: directed differentiation of pluripotent stem cells. Cell...PubMed PMID: 19255591. 17. Spence, J.R., et al., Directed differentiation of human pluripotent stem cells...PMCID: PMC3192723. 21. Grigoriadis, A.E., et al., Directed differentiation of hematopoietic precursors and...

...is located directly upstream of the fluorescent protein genes, Cre recombination is required before fluorescence... (XFP) of different colors to label an organ or entire organism. Because each cell can have multiple copies...transgene that will recombine independently, cells may acquire one of a variety of colors when a combination of...expressed. Each cell remains the same color for its entire lifetime and daughter cells retain the same color...tandem with a single promoter, but only the gene directly following the promoter is expressed. Cre-mediated...downstream in the construct. As RFP and OFP are directly downstream of the promoter in Brainbow 1.0 and...expressed in a single cell maintains its color over the entire length of the cell, even axons of up to 100 microns...

... professors how they hire scientists for their labs. Tom Ellis selects new hires from a pool of CVs collected...three lab heads how they go about selecting new hires. This is followed by some perhaps surprising, yet...for both parties, he will invite the prospective hire to the lab to see how they fit in with the team....are dedicated and interested in science. “Postdoc hires are simpler because of their documented history ...with their interests.” George Church selects new hires initially based on those who notice and embrace ...technology development, etc). Second, the potential hire visits the lab for a full day, gives a 1 hour talk... typically offer more flexibility in choosing direction of a project. If you expect more 1:1 time with...

...CRISPR/Cas9-introduced breaks are repaired. The following factors are required for NHEJ repair regardless of...pair of DNA ends together, forming a paired end complex The paired end complex then ligates compatible ...Non-Homologous End Joining (NHEJ) and Homology Directed Repair (HDR) – and how these pathways work, as...a higher capacity for repair, as there is no requirement for a repair template (sister chromatid or homologue...ends together. Similarly, it may be possible to direct insertion of an exogenous DNA fragment at a Cas9... structure of broken ends impacts how they are repaired. He works in Dale’s lab.        Dale Ramsden ...Blog Posts from Addgene: CRISPR 101: Homology Directed Repair CRISPR Protocol for Genomic Deletions...

...applications that require minimal cellular perturbation. Also, this method requires live, intact cells...attB-attP pair is aligned (sites facing the same direction) on a register, then the result of their recombination...attB-attP pair is anti-aligned (sites facing opposite directions) on a register, then the result of their recombination...sequencing is simple and the most accurate method as it directly reads the state of a register. However, each Sanger...difficult to isolate single registers or if it requires reading so many registers that Sanger sequencing...backbone) contains the register. Depending on the desired application, the inducible promoters we used to... be replaced by sensors that correspond to the desired inputs. Such sensors do not necessarily have to...

... allow for further purification, if desired, but it's not required for their use. Each RANbody is detectable...are a non-renewable reagent - Their production requires animal sacrifice and because they are an animal... for a summary. HRP HRP tagged RANbodies don’t require a secondary antibody for detection. Instead they...multi-labeling immunohistochemical protocols where either a directly labeled primary or a primary/labeled secondary...While Spaghetti Monster RANbody detection with a directly labeled anti-HA or -MYC antibody is less sensitive...synaptic partners (GRASP) assays No antibody required for detection RANbody is detected enzymatically...secondary Less sensitive than HRP-RANbody staining Requires secondary antibody staining HA Tagged GFP, RFP...

... for exclusively less-than comparisons. Paired and unpaired t-tests Samples that come from the same subject...You’ve acquired your long-awaited data – and it looks great! There are some exciting trends in your experimental...certain information about. The assumptions and requirements that need to be met for a parametric test are... - Quantitative If your data doesn’t fit the requirements above, a non-parametric test may be the way ...only care if one group is different in a certain direction (i.e., you just want to measure if a sample is...treatment, for example - should be analyzed with a paired t-test, because they eliminate subject-to-subject... on the other hand, should be analyzed with an unpaired t-test, which does account for subject-to-subject...

...component required (e.g. dsODN), doesn't require high read counts (fast multiplexing) Requires large cell...genome for all DSBs, doesn't require high read counts, fast multiplexing Requires delivery of dsODN (potentially...length of the gRNA given the stricter sequence requirement at the terminal 3’ PAM site (Fu et al. 2013; ...by sequencing (GUIDE-seq) (Tsai et al. 2015) and direct in situ breaks labeling, enrichment on streptavidin...BLESS detect double-stranded breaks and do not require high sequencing read counts making them fast and...contains both the rare off target event and the desired edit is low. Therefore, unbiased sequencing may...selected. Conversely, translational research may require the rigor of both forms of off-target analysis ...

...editing systems; first, RNA editing doesn't require homology-directed repair (HDR) machinery, and could thus...in non-dividing cells. Cas13 enzymes also don’t require a PAM sequence at the target locus, making them... sequence, but orthologs like LwaCas13a do not require a specific PFS. Cas13 enzymes do not contain the...domains responsible for DNA cleavage, so they cannot directly edit the genome. A Cas13-based RNA editing system...enzyme for further studies. PspCas13b does not require a PFS, and it is sensitive to mismatches in target...the lowest amount of off-target editing. In one direct transcriptome-wide comparison, REPAIRv2 reduced...activity. They then performed sixteen rounds of directed evolution across the full ADAR2DD to further optimize...

...Klompe et al., 2019; Strecker et al., 2019), which direct RNA-guided transposition. By natively combining...I-F CRISPR-Cas system. The four major components required for RNA-guided DNA integration are (Fig. 1A, top...transposition in type I-F INTEGRATE. The requirement for multiple Cas proteins to form the Cascade...targeting (Halpin-Healy et al., 2020). Genomic targets require an upstream 2-bp PAM sequence for recognition by...instead of three Cas proteins (Fig. 1A, bottom), and require only two transposase proteins (TnsB and C). While...effector plasmid (pEffector) expresses all seven required genes and the CRISPR RNA under the same promoter...: Clone spacer(s) into the CRISPR array Clone desired DNA cargo into the mini-transposon Transform plasmid...

...fluorescent donor to your protein is to hardwire the molecule directly into the protein’s peptide sequence...(the donor) is excited, it can transfer energy directly via resonance to the second fluorophore (the acceptor...translation (Ermolenko et al., 2007). All that is required is that the proteins/nucleic acids/lipids of interest...delivered to the right place? FRET experiments require targeting the donor and acceptor fluorophores to...experimental design is that the targeting specificity requirements are different for the donor and acceptor fluorophores...codon (often amber i.e. UAG) engineered into a desired site. During translation, the novel tRNA/unnatural...overhaul of the cell’s native tRNA synthetases is required and thus far, this method has proven most effective...

... ori and requires the host proteins DnaA and IHF to initiate replication. But pSC101 requires a third ...RepA binds to directly repeated sequences called iterons located near the ori and directly interacts with...said to be under relaxed control, as they only require the host's replication machinery for elongation...have enough nutrients available to complete the entire round of replication. Initiation is the primary...versions, but binding of the low-affinity versions is required to start the replication process. When enough ...creates two replication forks that move in opposite directions away from the ori. In the elongation phase, a...on host-encoded proteins, it can be regulated indirectly via the same mechanisms that affect DnaA and ...

...is necessary to be direct and clear. There is place in human discourse for indirect language, but it is...Giving timely and useful feedback is an absolutely required aspect of being a successful manager. Great bosses...over, what would you do differently? Beware of indirect language You must be honest about performance ... should be aware that they are at risk of being fired and should be looking for a new position. One of... mistakes I see is managers who hint at or use indirect language to talk about real performance issues...issues. Watch this little video to learn more about indirect language from Steven Pinker. As a manager you ...Scientists. Subscribe to the Addgene Blog to follow the entire series as it is published. For more reading try...

...practices, their opinions towards ELNs, and their desired functionalities i.e. main priorities of different..., and the fact that long-term use is likely to require on-going maintenance and support.” Besides that...it as very important. One comment reflected the desire for a flexible generic solution, rather than an...switching from paper to an electronic lab notebook requires time and knowledge. In a recent article, we translated...reporting on a project – printing your data out can be required every once in a while, so it is good to make sure...your lab need to meet any specific compliance requirements – e.g. 21 CFR part 11, HIPAA? Answering this... that take any ownership over your data or have direct access to it. Your data should be kept private ...

... for a site-directed mutagenesis PCR that uses mutagenic primers to introduce all desired codon changes...generation of a plasmid library that expresses all desired variants of a protein. Applying a selective pressure...phage-derived uracil labeled template, this method requires further DNA shuffling to combine pairs of mutations...of your liking. This method is similar to site-directed mutagenesis (SDM) PCR, but with a twist since ...the plasmid. Again, if they are in the opposite direction, both strands of the plasmid will be nicked during...of your plasmid. If the sites are in opposite directions, you just need to use a plasmid with BbvCI restriction... the gene or use custom gene synthesis or site directed mutagenesis to remove the BbvCI site(s). Overall...

...but this approach would require robust stem cell editing to maintain the desired phenotype. Precision genome...skipping. Addgene depositor Charles Gersbach used paired zinc finger nucleases to remove exon 51 in DMD ...For such a therapy to be successful, multiple requirements must be met. First, CRISPR would need to be ...especially well suited for genome editing. The homology-directed repair (HDR) pathway is downregulated in mature...is maintained for at least six months. Future directions and obstacles to clinical CRISPR editing Given...NHEJ-mediated editing, but most other diseases will require HDR-mediated precision editing tailored to smaller...:10.1126/science.aae0169 2. Long C, Amoasii L, Mireault AA, McAnally JR, Li H, Sanchez-Ortiz E, Bhattacharyya...