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...clone out of 104 to 105 colonies. Pro Tip: Use a high-fidelity Taq polymerase with proof-reading ability... center of the sequence. Since lambda red has a higher recombination frequency when the lagging strand...strands. One of the two oligos will recombine with a higher efficiency than the other and you’ll find out empirically...frequency between 0.1% to 1%, and can be increased to as high as 25-50% by designing oligos that avoid activating...deletion of >20 nucleotides 1 out of 104 to 105 Use a high fidelity Taq polymerase to generate insert ssDNA...these strains, expression of exo, beta and gam is tightly regulated by the endogenous phage promoter pL and... allows for a mobile recombineering system, but tight regulation of expression is required for a successful...

...while being accessible simply by dialing up the right nucleic acid sequence (Addgene can help with this...diverse kinds of proteins that one finds in cells. High yield of reaction — generating product without a... a lot of left-over starting components. High rate constant— time is money and things move fast in biology...good enough if the reacting parts are present at high concentrations, but in biology one often works with...situations. Specificity — coupling must happen in the right place in living systems when there are lots of other....1c00469  Howarth, M. R. (2025). Click biology highlights the opportunities from reliable biological reactions...

...significantly reduce costs of producing plasmids with high-throughput methods that save on reagents, consumables...many labs are quite happy to share, labs that get high volumes of plasmid requests can end up refusing ...traditional PCR/digest/ligate methods and picking the right one can save you time if you’re working at speed... that works for your experiment. Make your work “high-throughput” Instead of making one plasmid at a time...t scale or have limited scaling. The downside of high-throughput work is that you give up a lot of flexibility...minute experiments or changes. Fun fact: Addgene’s high-throughput approach means we virtually always use... Addgene Blog 5 factors to help you choose the right cloning method Find blog posts about scientific ...

...conformational state may change. If your protein might become slightly denatured during your experimental protocol... or a goat) is injected with an immunogen. This might be the full-length protein that you want to generate...that recognize your target protein without causing high background due to cross-reactivity. When to use ... in many labs. Polyclonal antibodies also have a high sensitivity to the target protein due to their ability...or recombinant antibody instead. Otherwise, you might experience variability in the strength of staining...strength. In general, polyclonal antibodies produce a higher level of background noise compared to monoclonal...

...abilities of the lesser-known Cas enzymes while also highlighting their similarities and differences to Cas9. ...therapeutic delivery.   Cas14 – Mini, but single strand mighty Unlike many other Cas enzymes, Cas14 targets ssDNA...ssDNA instead of dsDNA. It cleaves ssDNA with high fidelity - sensitive to even a single mismatch in the...sequence. Why would you want to give it a go? The high specificity of Cas14 makes it a promising detection... Fast facts – PAM requirement: none. Best for: high fidelity ssDNA targeting. RNA targeting Cas enzymes...enzymes are worth taking a look at. Choosing the right Cas can really put your experiments Nsync.   References...

...given problem, Empowers the writer, And offers insight into a career as a Professor. Step 1: Your ...time to review the literature and offer educated insight into what the desirable features of future technologies...field. Research strategy The Research Strategy highlights i) the significance and innovation of the project...statements. While some of the statements may be slightly far reaching, remember that you are writing a ...text. Specific aims The Specific Aims section highlights each milestone that must be met in order to deem...

...basics of the technology. In this blog, we will highlight some common MoClo applications and kits. Keep ...beyond what we discuss. If you can think it, MoClo might help you achieve it! MoClo applications and specialized...cassettes (Shaw et al., 2023). Those looking to express high levels of their gene of interest in yeast should...10.1021/acssynbio.6b00337. Lee, Michael E., et al. “A Highly Characterized Yeast Toolkit for Modular, Multipart...s12870-020-02388-2. Stuttmann, Johannes, et al. “Highly Efficient Multiplex Editing: One‐shot Generation...doi.org/10.1111/tpj.15197. Grützner, Ramona, et al. “High-Efficiency Genome Editing in Plants Mediated by ... Addgene blog Plasmids 101: Golden Gate Cloning High Complexity Golden Gate Cloning with NEB’s DAD New...

... (TMP-tag) (Miller et al., 2005). Although this tight interaction is not covalent, later work showed that...systems can be complicated to implement, they can be highly specific with a small and minimally-disruptive ...  Applications Organic dyes are typically both brighter and more photostable than fluorescent proteins...in a new fluorescent ligand and see the results right away. Or perform pulse-chase experiments by sequentially...membrane. So, you should investigate your options and weigh the potential benefits and costs before investing...investing in one approach or the other.   Finding the right tag for you Protein-based tags typically provide...the fluorophore’s microenvironment to enhance brightness, stability, or other properties is an active ...

...Left) The light chain and heavy chain genes of the antibody are encoded in a plasmid. (Right) An assembled...If you were born before 1985, you might remember going to the store and buying CDs when you wanted to...cell line. The genes encode the heavy chain and light chain for the antibody and when translated into ... reagents. These include conventional heavy and light chain recombinant monoclonal antibodies as well ...encoding antibodies, scientists will be able to make higher affinity antibodies, modify antibody function, ...10.1080/19420862.2018.1445456 Crosnier C, Staudt N, Wright GJ (2010) A rapid and scalable method for selecting...

...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...be easily visualized.  The tool-kit consists of highly useful vectors compatible with Gibson and Saptrap...CRISPR corner By Jennifer Tsang Here are a few highlights from recent CRISPR plasmids. To find all of the...Rex's lab.  vfCRISPR, or very fast CRISPR, is a light-inducible CRISPR system developed by Taekjip Ha’...target DNA without cleavage until it's activated by light. Once activated, Cas9 cleavage can induce DSBs within...

...about your prior research. This is a chance to highlight your prior accomplishments and demonstrate your...skills that make you qualified for the position. Highlight the outcomes or deliverables of your work, such...extraversion for me. Make sure to get enough sleep the night before. Have your morning coffee, if that’s part...part of your daily routine. Stay hydrated. You might have lunch on your schedule, but it will probably be...’t have technical challenges, your interviewers might.  You’ll probably still be required to give a job...

...Although other methods have been able to achieve much higher activation, dCas9-VP64 is great for experiments...targeting single genes, SAM consistently shows the highest levels of gene activation compared to other CRISPR...Performance Generally, VPR is found to have significantly higher activation levels than the initial dCas9-VP64 activator...rearranging chromatin structure. This method has shown higher activation levels than SAM in some loci across ...has been shown to activate genes at 2- to 3-fold higher than SAM, SunTag, and VPR (Zhou et al., 2018). ..., Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient Cas9-mediated transcriptional programming...https://doi.org/10.1038/nmeth.3871 Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena ...

...post was contributed by guest blogger Sean Mac Fhearraigh, co-founder of www.assaygenie.com. No matter ...required and no one lab or professor becomes an overnight success. Below I have detailed some tips to improve...in different fields that can provide different insight into an area - not just working within the established...and possibly a new perspective on the field. 3. Highlight your network and your collaborators It’s a good...collaborators can also help you get some critical insight into what you're doing wrong. All this helps to...  Many thanks to our Guest Blogger, Sean Mac Fhearraigh! Seán was a PhD student at University College...

...current findings in nucleic acid science. I was delighted to see that the majority of attendees were scientists... enjoys preferential governmental policies for a high-tech development zone so many science and technology...fauna. I tried to find out what certain ingredients might be used for (like what would one take dried seahorse...blot results and we didn’t give tRNAs much thought. High throughput transcriptome sequencing methods including...families and provide a way to guide inheritance that might be less error prone than DNA replication. He too...things.” He further cautioned the distinction of “higher” and “lower” life forms as we shared millions of...the bacteria sensitive to known antibotics. These might be used on hospital surfaces and in hand sanitizers...

...flow cytometry During conventional flow cytometry, light emitted by a fluorophore is guided to a single detector...on the other hand, utilizes the full spectrum of light to distinguish one fluorophore from another. Instead...upwards of 30-40 colors), spectral flow cytometry might be the better choice. Of note, there is no minimum...of the markers you are including in your panel. Highly expressed markers should be paired with dimmer ...markers with low expression should be paired with brighter dyes, so they can more easily be detected. Cytek...cells when designing your panel. If you have two highly overlapping cellular markers, it is important to...on the Addgene blog Antibodies 101:Selecting the Right AntibodyAntibodies 101: Secondary AntibodiesAntibodies...

...should be easy, right? Yes and no. If you’re just trying to create a knockout, you might be happy targeting...near your insertion site, the corresponding gRNA might be undesirable due to potential off-target sites...they saw that xCas9 3.7 cleaves multiple PAMs at a higher efficiency than SpCas9: NGT (4.5-fold efficiency...genome-wide off-targets, since increasing PAM flexibility might also increase off-target activity. Surprisingly,...generation cytidine base editor architecture (BE3) slightly improves NGG PAM editing efficiency over that ...Evolved Cas9 variants with broad PAM compatibility and high DNA specificity." Nature (2018) doi: 10.1038/nature26155...

...daytime. They mostly stop emerging at night. An incubator with a light cycle makes for very predictable work...temperatures like 18°C, flies take longer to develop. At high temperatures like 30°C, flies develop faster but...When flies first emerge, their bodies will be very light in coloration and can look very plump. On the side...flies. Like Addgene, the Drosophila community is highly sharing-oriented, and there are many resources ...your fly gene using the search box in the upper right hand corner. They have video tutorials under the...useful information on topics such as choosing the right balancer chromosomes to use. If you’re not ready...

...False, width='600', height='189', player_id='31668202421', style='text-align: right;' %} Figure 3. An...data with a 7-day rolling mean gave us a crude insight into the magnitude of the effect COVID-19 had on...there are many new COVID-19 cases being reported (right, red), likely due to quarantining {% video_player...most impacted or least impacted. These results highlight the instability and uncertainty in estimating ...methods for fundamental scientific discovery and high-impact applications.       Alvin Hsu is a graduate...

...outperformed another CRISPR library with 6 sgRNAS, highlighting the benefit of sgRNA design in CRISPR libraries...CRISPRi sgRNA library While CRISPRko libraries are highly effective at identifying strong hits in genetic...because it's essential for viability. In addition, high copy number genes may mimic viability genes as they...knock-out library while mitigating toxicity induced by high copy number dsDNA cutting. A Dolcetto library containing...wide CRISPRko, CRISPRi, and CRISPRa libraries are highly effective for probing gene function while using... 1. Doench, John G., et al. "Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation...

...a new coordinator for the annual Math & Science Night, I took the reigns. I also volunteered when our ... started coordinating the annual Math & Science Night, a chance for parents and other members of the community...activities. If your school doesn’t have a Math & Science Night (or some equivalent), here are some quick tips to...activities we had for my school’s Math & Science Night. There are plenty of places to get ideas for science...for all of these questions, and for any topic you might present to middle schoolers, is to provide current...again next year. There are many reasons why you might want to reach out to your school community as a ... Elementary for trusting me with Math & Science Night, and a special thanks to Joanne Marks, Jonathan ...