...high-throughput multiparameter screen (Koveal et al. 2022). Lactate is a key metabolite in glycolysis, ...of year approaching. We’re always looking for something to brighten our day, whether it’s memories of ...extracellular oxytocin dynamics in the brain. Nat Methods. 2022 Sep 22. pii: 10.1038/s41592-022-01597-x. ...screen to select variants based on performance metrics specifically for two-photon microscopy. The resulting...valuable tool to study cellular physiology and metabolism under various conditions. LiLac has improved ... here!   Koveal D, et al. A high-throughput multiparameter screen for accelerated development and optimization...Temperature Imaging with a Genetically Encoded Nanothermometer. Nano Lett. 2022 Jul 27;22(14):5698-5707. doi...

... multiple of three will do. A popular screening method for knockouts is Tracking of Indels by Decomposition...generating a knock-in mutation, the best screening method depends on what your desired edit is. Large knock-ins...knock-ins Are you introducing a large insertion, something 20 base pairs or bigger? Then screening by PCR...indicating targeted edits.   Interested in this method, but your edit doesn’t affect any endogenous restriction...be a marker for edited cells. As with all other methods, you’ll need to further validate the specific mutations...using sequencing confirmation. All knock-ins A method applicable to knock-ins both large and small is... sequencing trace file from the donor DNA. This method can precisely estimate editing frequency in a bulk...

...Looger LL, Simpson JH. Nature Methods. 2011 Mar;8(3):253-9. doi: 10.1038/nmeth.1566. Epub 2011 Feb 6. PubMed...Dickson BJ, Salecker I. Nature Methods. 2011 Mar;8(3):260-6. doi: 10.1038/nmeth.1567. Epub 2011 Feb 6. PubMed...Lichtman JW, Sanes JR. Nature Methods. 2013 May 5:10(6):540-7. doi: 10.1038/nmeth.2450. Epub 2013 May 5. PubMed...and the neuromuscular junction. In zebrafish, the method has become very useful in lineage tracing; “Zebrabow...homeostasis results from neutral competition between symmetrically dividing Lgr5 stem cells. Snippert HJ, van der...

...able to understand what metrics would be useful and how to organize the metrics in a digestible manner....building strong connections with coworkers and doing something meaningful with my career. I can’t pinpoint...summer travel plans while eating lunch. There was something so special about her pure interest and curiosity...biweekly report of our social media analytics, something that had never been done at Addgene before. This... and trampoline all at once. I was able to do something that was challenging while helping us share our...confident in myself, my abilities, and my future - something I didn’t expect from a co-op experience.  Sarah...

...described thermodynamics-based methods of serotype confirmation, this method does not require any additional... and use cases in Human Gene Therapy. Existing methods for AAV quality control Historically, AAV vector...sequencing, TA-based ligation and tagmentation-based methods for AAV genome sequencing (Tai 2019; Lecomte 2015...1972). Using qPCR and restriction enzyme based methods, we showed that the DNA extract is a heterogeneous...Leenay R, May AP, Paulk NK (2019) Fast-Seq: A Simple Method for Rapid and Inexpensive Validation of Packaged...Genomes in Academic Settings. Human Gene Therapy Methods 30:195–205 . https://doi.org/10.1089/hgtb.2019.110...

.... Nature Methods. 2018. PMID: 29735997 bioRxiv preprint posted July 4, 2017 A new method to identify...quantitative analysis using different detection methods. Since the anti-GFP nanobody is able to bind fluorescent...PNAS 2018. PubMed PMID: 29915061.        A new method to identify proteins occupying a specific genomic...investigated protein binding at the TERT gene, but this method enables the unbiased discovery of proteins involved...biotin-labeled proteins can them be analyzed by mass spectrometry allowing a researcher to specifically identify...the C-BERST plasmids here. Gao at al. Nature Methods (2018) Pubmed PMID: 29735996.   ...

...selection-based CRISPR/Cas9 method to generate knock-ins on any genetic background. Their new method uses a selection...Reiner DJ, Goldstein B. Nat Methods. 2013 Oct;10(10):1028-34. doi: 10.1038/nmeth.2641. Epub 2013 Sep 1. PubMed...papers describing CRISPR/Cas9 genome engineering methods for C. elegans were released, signaling a new era...modifications, is very robust in C. elegans, and the methods for HDR-mediated modification continue to be improved...PCR-based screening to detect mutants, but this method limits screening to a few hundred worms, making...

...about experimental methods and approaches. It’s very easy to think that the methods section of a paper...Press we developed STAR Methods to improve the structure and transparency of methods sections, including ...was not, and it was just that they were doing something differently. Communication comes in many forms—from... researchers can use them, to making sure that methods and protocols are available and detailed enough... of other Cell Press journals, including Cell Metabolism, Cell Reports and Joule. In September 2017 she...

...the context of cloning, DNA assembly refers to a method of physically joining multiple fragments of DNA...synthetically designed DNA sequence. There are multiple methods of DNA assembly available, including: Gibson Assembly...assembly. Finally, the NEBuilder® HiFi DNA Assembly method from New England Biolabs® has minimal upfront requirements...assemblies. Overview of NEBuilder HiFi DNA assembly method As with any well-executed cloning endeavor, NEBuilder...NEBuilder requires upfront planning. This cloning methodology requires that the fragments to be joined contain...complex DNA assemblies and improvements in these methodologies offer streamlined and simplified alternatives...Assembly and Other Long-Homology Based Cloning Methods Screen Plasmids by Colony PCR Addtional Resources...

...Challenges of existing mutagenesis methods Several continuous diversification methods exist, but there are a few...diverse libraries of genetic variants. Several methods for generating mutant libraries exist, but none...EvolvR, a CRISPR-Cas9 based targeted mutagenesis method developed by the Dueber Lab at Berkeley, provides... the Phage Assisted Continuous Evolution (PACE) method requires constant turbidity of the culture, so ... the Bassik Lab and the Chang Lab have created methods that introduce diversification by targeting a site... on the Addgene blog Learn about other CRISPR methods for bacterial genome engineering   Learn more about...

...Plasmids 101, we can talk about an even more specific method of screening your cloning reaction. Being able ...an insert? Blue-white selection is a widely used method to do just that! (To read about other screening...thus should be used in conjunction with selection methods. Combining selection and screening ensures that...blue-white screening). Other types of screening methods Although blue-white screening is probably the most...plasmids containing an insert, there are other methods. Positive selection vectors encode a gene which...selection is also used in conjunction with this method to ensure that positive colonies do have the plasmid...commonly used in Gateway® Cloning. There are also methods to select for plasmid-containing cells without ...

...the “elegans” methods outlined in this post – particularly the use of co-selection methods – could greatly...templates in C. elegans. Methods 121–122:86–93 . https://doi.org/10.1016/j.ymeth.2017.03.023 Paix A, Wang...exhibit complete embryonic lethality at 25 ºC. This method resulted in efficiencies ranging from 11-100% of...selection allowed me to optimize a range of editing parameters: oligo repair templates with homology arms of...years:  Repair template design The Meyer lab meticulously characterized several aspects of editing. First...Cas9 Since the development of CRISPR/Cas9 editing methods, other Cas proteins have been adapted for use in...

...O, Zhang F. Nat Methods. 2014 Aug;11(8):783-4. doi: 10.1038/nmeth.3047. 10.1038/nmeth.3047. PubMed PMID...add 3 microliters of 5 mM star anise?” This is something I was expecting when I set out to make my first...obtained through Addgene’s viral service and the methods sections of scientific articles (gasp). To my delight...the duplex and run it as an undigested control. Sometimes the PCR products can look smeared, so having an... not (Figure 4). 5. In theory you could use densitometric analysis to quantitate the intensity of the ...knockout a single allele in a human line, using methods based on homologous recombination (Rago et al.,...

...Aveiro, Portugal. If cancer was a musician, then metastasize is touring. But you know what a touring musician...divided into 3 phases: initiation, progression, and metastasis. The microenvironment’s physiology is deeply ...the tumor. How do microenvironment enzymes and metabolic reprograming influence tumors development? Being... vice versa, let’s take a look at cancer cell metabolism. Cancer cells promote oxidative stress leading...altered fibroblasts and promote the development and metastasis of tumors through inciting inflammation. Speaking...of cells affect tumor progression, enzymes and metabolism reprogramming aren’t the whole story. It’s essential...Tumour exosome integrins determine organotropic metastasis." Nature 527.7578 (2015): 329. PubMed PMID: 26524530...

...He has recently developed a third-generation intensiometric glutamate sensing fluorescent reporter (iGluSnFR3...biosensors or biosensors that detect new redox metabolites are vital for researchers to better understand...withdrawal before they occur.  First, she used a genetic method to tag neurons activated by opioid withdrawal with... opioid withdrawal, providing insight into new methods for treating patients in recovery from opioid use...is in progress, she has validated the dual virus method to study CR neurons which are depicted here. (A...biosensors based on their dynamic properties. Her method takes a pooled library of mutant sensors expressed...engineer many types of biosensors.   This screening method allowed Tian to optimize a red‑shifted archaerhodopsin‑derived...

.... However, CNO is back-metabolized to clozapine and other clozapine metabolites and these can have off-target...concentrations of clozapine N ‐oxide (CNO). CNO is a metabolite of the antipsychotic clozapine and seems to be...experiments in this species may be entirely due to back-metabolism of CNO to clozapine, demonstrating the need for...activity, and do not seem to have the same back-metabolism issues as CNO and are attractive alternatives...2004). The highly efficacious actions of N-desmethylclozapine at muscarinic receptors are unique and not...

...topic.  Meta Meta is a literature discovery platform developed by the Chan Zuckerberg Initiative. Meta is ...number of manuscripts that cite the background, methods, or results section is also displayed. Saving ...interest. You can also just follow feeds curated by Meta. Feeds automatically update as papers are published...number of times a paper is shared online, or its altmetric score, is also displayed in your feed. By clicking...whole.   Saving papers Papers are easily saved to a Meta library. The library will suggest additional papers...

...of microbial opsins. Nature methods. 2012;9(2):159-72. doi: 10.1038/nmeth.1808. PMID: 22179551; PMCID:...run it? And, most importantly, what are the methodological and experimental limitations and considerations...considerations you may not find in the materials and methods sections of optogenetics papers. Optogenetics...Delivering your opsin  The standard opsin delivery method is local injection of an adeno-associated viral...may need to adjust coordinates based on your own method of identifying bregma but it is best to spend the...stimulation. Testing opsin expression  Regardless of the method of expression of your opsin gene, it is essential...dissection of neural circuitry function. Nature methods. 2011;8(9):745-52. PMID: 21985008; PMCID: PMC3191888...

...neuronal tracing methods Traditional tracing compounds either produce a colorimetric product or are fluorescent...technology, new methods have been developed to study neuronal connectivity. These methods are less neurotoxic...neuroscience methods such as electrophysiological recordings. The origins of these methods are naturally...inserted electrodes) may be applied, but these methods can have a negative impact on cellular health. ...versus Intradiaphragmatic Injection. J Neurosci Methods 241, 137–145. PubMed PMID: 25555356. PubMed Central...for single- and double-label studies. J Neurosci Methods 55, 65–78. PubMed PMID: 1381034. Watakabe, A., ...

...as the method is dependent on number of samples rather than number of cycles.  Choosing a method  As with...real-time PCR. J Microbiol Methods. 2006 Jun;65(3):476-87. doi: 10.1016/j.mimet.2005.09.007. Epub 2005 Oct...confidence in the results you need.  Whichever method you choose, resist the temptation to over interpret...Copy Number Variation with Droplet Digital PCR. Methods Mol Biol. 2018;1768:143-160. doi: 10.1007/978-1...quantitative real-time polymerase chain reaction methods for the determination of amplification of Plasmodium...