...shared his flow cytometry expertise in three posts. An Introduction to Gating in Flow Cytometry, Designing...shared her flow cytometry expertise in two posts. Flow Compensation and Flow Cytometry Controls Priyamvada... guide to understanding your flow data. Flow Cytometry Readouts Toj, a summer intern who shared their...validation expertise. Antibody Validation for Flow Cytometry Rob Hurt, whose expertise and suggestions were...

...scientists are no exception to this effect -- meticulously designed experiments in labs around the world...modification also eases the interpretation of the parameters we infer, since scientific productivity is better... model to give uncertainty estimates for each parameter, we added log-normal noise to each of the terms...gave us the maximum likelihood estimates of each parameter. We approximated the data likelihood with multivariate...dates impacted by each effect. The inferred parameters suggest that COVID-19 induced a 2x–5x reduction...the instability and uncertainty in estimating parameters from relatively sparse data from China. An important... uses applied machine learning and statistical methods for fundamental scientific discovery and high-impact...

...settings: The sense of being the first to discover something. The thrill of having an experiment yield clear...if I was asked that question today, because I sometimes wish for a stretch of time that doesn’t involve...mentally challenging than lab work at times.” Sometimes a desk job is defined as sitting down at a computer... between tubes, look through microscopes, and sometimes you even wear a lab coat! These are the things...is that I’ve never been bored. There is always something to do and there is no thumb twiddling. But you...time to come to realize that meetings were not something that took me away from my work. The meetings often...share their knowledge with the community (and sometimes conduct their own independent research!). There...

...-mutant rabies virus. Nat Methods 4:47–49 . https://doi.org/10.1038/nmeth999 Wickersham IR, Lyon DC, Barnard...by nonspecific complement mechanisms in serum. Sometimes this is due to the cells that the viral vectors...Scientists have been using similar pseudotyping methods with other viruses in a BSL-2 lab instead of a ...the upregulation of LDLR expression using other methods.  In terms of preparation, VSV-G is stable and ...given brain region (Mazarakis et al., 2001).  This method can also allow scientists to trace a neuron’s projections...Vectors: One Vector, Many Guises. Human Gene Therapy Methods 28:291–301 . https://doi.org/10.1089/hgtb.2017.084...

...genders, educational, and professional backgrounds. Sometimes this can lead to wildly different ideas of how...our work.  Agile at Addgene We follow an agile methodology of software development called Scrum. The Scrum...during checkout. Demo details for this would be something along the lines of showing that the checkout takes... decide how to best approach it in our code.  Sometimes while thinking through these steps, we discover...more in-depth knowledge of one area, and when something urgent comes up, that person is the first in line...In some parts of our code, we might be calling something a sample and in other places, we might be calling...clean, reusable code when working on a feature, sometimes we have to take short cuts or add a band-aid to...

...target metabolite creates an aptamer that will only bind to DFHBI in the presence of that metabolite (Fig... led by Jack Szostak and Larry Gold, developed methods for selecting and amplifying RNA sequences that...including proteins, peptides, amino acids, drugs, metal ions, and cells (even pathogenic bacteria). That...to incorporate their target molecule, such as a metal ion, or fold to incorporate themselves into a larger...difficult-to-study RNA-modifying enzymes, such as the RNA demethylases FTO and ALKBH5. Their ease of production have...2. Baker, Monya. “RNA imaging in situ”. Nature Methods 9, 787–790 (2012). 3. Filonov, Gregory S., et al...

...CRISPR-Cas9 genome engineering parameters via a library-on-library approach. Nat Methods 12:823–826 . https://doi.org...transcriptional programming. Nat Methods 12:326–328 . https://doi.org/10.1038/nmeth.3312 Chavez A, Tuttle M, ... in multiple species. Nat Methods 13:563–567 . https://doi.org/10.1038/nmeth.3871 Gilbert LA, Horlbeck.../doi.org/10.1038/nmeth.3473 Chavez A, Scheiman J, Vora S, Pruitt BW, Tuttle M, P R Iyer E, Lin S, Kiani...

...isolated from a methylation competent bacterial strain (e.g. DH5α which is dam+). Methylation deficient bacterial...and Daniel R. Marshak. "Dimethyl sulfoxide-mediated primer Tm reduction: a method for analyzing the role...template. The parent template is removed using a methylation-dependent endonuclease (i.e. DpnI), and bacteria... of the primers. Because you will be using a methylation-dependent enzyme (DpnI) to eliminate the parent...is unique in that it cleaves only DNA that is methylated at the adenosine of the GATC recognition site...

...fluorescently labeled probes. Colorimetric and luminescence based detection methods are also commonly used. ...with fluorescently labeled ones allowing for fluorometric-based detection. This increased sequencing speed...sequencing machines (9). Today, similar technologies, sometimes referred to as sequence DNA as it synthesized ...fluorescently labeled nucleotides are incorporated, a method called pyrosequencing. The drive for faster, higher...typically rely on Sanger sequencing but instead use methods like pyrosequencing that are parallelized to sequence...of sequencing should bring efficient, low cost methods for sequencing large amounts of DNA quickly and...

...microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 References Abreu, N...microbial opsins. Nature Methods, 9(2), 159–172. https://doi.org/10.1038/nmeth.1808 Nagel, G., Szellas,...multiple intra-cellular signaling cascades. Optical methods for controlling GPCR activation have been developed... than is achieved by traditional pharmacologic methods. A number of strategies have been developed for...mechanisms that are often cell-type specific. If it is something you’re considering for your experiment, note that...

...proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody...western blot, or western, is one of the simplest methods to detect the presence or absence of a protein ...proteins typically do not transfer as well using this method.  Finally, commercially available dry blotting ...antibody, consider reviewing the materials and methods sections of those publications.  Finally, always...quantification of a protein, consider using an alternative method such as an enzyme-linked immunosorbent assay (ELISA...modifications such as phosphorylation, acetylation, methylation and ubiquitination. For example, to test if a...

...Staphylococcus aureus (SaCas9) . AAV is also a promising method for gene therapy. Of the commonly used viruses,...limits the packaging capacity of AAV to 2.4 kb. Methods to Increase Packaging Capacity: To increase the...full-length transgene can then be expressed. This method allows for longer transgene expression, but expression...capacity depends on homologous recombination. In this method, a gene is divided between two transfer plasmids...efficiency (<1% of wild type). If either of these methods can be made more efficient, the use of AAV viruses...

...the trick is to utilize a second immobilization method. One possibility is covalent modification of the...a similar function as GST in that it provides a method to express a foreign protein in bacteria and subsequently...determine binding kinetics using a multitude of methods including surface plasmon resonance (Biacore, GE... GE Life Sciences) or bio-layer interferometry (Octet, Pall ForteBio). Tips for using pMal-T-Avi-His/BirA...commercially available. Factor Xa loves calcium, but metal-chelate matrices, not so much. Maybe a calcium-independent...that have been biotinylated using less efficient methods. Many thanks to our guest blogger Jon Backstrom...

...gene, Mettl7a1, whose expression helped coax cells towards successful iEP reprogramming. MettI7a1 encodes...encodes an uncharacterized putative methyltransferase. Adding Mettl7a1 to the standard Foxa1-Hnf4α retroviral...CellTag CellTagging is a lentiviral based tagging method that labels cells with heritable barcodes. Barcodes...reprogramming and direct reprogramming Explore different methods for delivery of reprogramming factors for generating...

...launch a product? Make an informed decision about something? Fix a problem? Test a hypothesis? Whether you...to make progress towards open-ended goals whose metrics for success are defined by ongoing mission fulfillment...The benefits of a team So why is it helpful, or sometimes even necessary, to build a team in the first place...diverse group of people to provide that base. Sometimes there’s just too much work If you are one of those... frustrating than being on a leaderless team. Sometimes the leader just has to be the person to say “OK... guys, we need to have a meeting about this.” Sometimes the common roles of a leader can be successfully...expertise your current team members don’t have - sometimes you just don’t know who you need until the team...

...validation of antibodies. Nat Methods 13:823–827. https://doi.org/10.1038/nmeth.3995  ...antibody-dependent approach. Antibody-independent methods may include analysis of transcriptomic or proteomic... between the levels of target detected in both methods. These approaches have relatively low sensitivity...be concerned that your antibody is binding to something unexpected. This approach has high specificity...epitopes of this protein.   You perform flow cytometry using both antibodies, as well as a third unrelated...sample using the antibody and then uses mass spectrometry to identify the captured proteins. If the antibody...

...Watch out for methylation issues. Enzymes like XbaI and ClaI are sensitive to methylation and their activity...sequence of all the plasmids we distribute. This method is time-intensive, so we recommend a variety of...sequence. Once you have purified plasmid DNA, this method can be done right in your lab in less than a day...will need to purify your DNA from a dcm or dam methylation-deficient bacterial strain such as JM110 or INV110...lanes look like your uncut lane then there is something wrong! Quantify your DNA. Loading too much DNA...

...development of multiple virus packaging methods. Across these methods, the following components are needed...Packaging using 293T cells This method is very similar to lentiviral packaging methods. The three components described...Packaging using helper-free packaging cell lines This method utilizes a packaging cell line that stably expresses... these genes in trans by specific plasmids. This method, therefore, reduces the number of plasmids that...

...mutated methionine was not enough to prevent alternative initiation, so they mutated the methionines at position...without any off target effects. However, these methods can be labor intensive and quite time consuming... will focus on two fluorescent CRISPR reporter methods, SRIRACCHA and GEMCherry2. We will cover single...start site initiation, the lab removed the native methionine start site. This first iteration was named GEmCherry1...for your CRISPR mutant of interest, give these methods a try.  References Højland Knudsen C, Ásgrímsdóttir...

...eukaryotic cell through non-viral methods including both chemical and physical methods in the lab. Chemicals like... the lab. Let’s take a look at these different methods of DNA insertion. Transformation Transformation...conjugation Conjugation was the first extensively studied method of gene transfer and was discovered in 1946 by ...Kostman.   Transfection Unlike the last three methods which can be used in prokaryotes, transfection ...the negatively charged cell membrane. Physical methods such as electroporation or microinjection actually...