...plates) (see box below). This initial transformation should be performed using a highly competent strain that...polymerase is a phage-derived polymerase that can perform whole genome amplification (by a mechanism known...certain modifications to DNA bases.  Ligate and transform your resultant plasmid libraries into chemically...chemically competent E. coli and analyze transformants for appropriately sized inserts. We consider a library...culture, dilute and plate the complete library of transformants for single colonies (this usually requires 10...extract plasmid DNA directly from the colonies. Transform the resultant plasmid DNA into E. coli BL21 (DE3... protein production), then dilute and plate transformants for isolated colonies (150-200 per plate) across...

...expression atlas, including over 3,500 human TF isoforms, to help researchers understand this regulatory...encodes approximately 1,800 TFs, with >3,500 unique isoforms transcribed from these sites. The Zhang lab generated...cultured following a differentiation protocol before performing single-cell RNA sequencing. The sequencing results...were differentiated and single cell RNA-seq was performed to map TFs to cell fates. (Adapted from Joung ...MORF to work Screens and selections In its current format, the MORF library is most immediately useful for...on cellular processes. For help with library transformation and amplification, refer to the Zhang lab’s...library to further investigate specific genes and TF isoforms. Due to the size of the collection, we’re releasing...

... them. That’s where our content - the useful information on scientific literature, technical skills, and...e-books – comes into play. Our goal is to provide informative content to help researchers in creating and sharing...spring of 2022. The first step was to understand information our guide needed to have. Through a series of...applications should be included and what basic information someone new to antibodies might need to successfully...primary literature search to collect up to date information on the scientific topic. If that’s not enough...from papers or image sharing repositories. Then we format, do a final review to catch those pesky typos, ...research community! We work to provide engaging and informative resources that answer questions our depositors...

...writers have been curious about the capability of transforming the genetic code of living organisms and its...that Streptococcus pneumoniae bacteria could be transformed from one organism to the other. This is now famed... myth that protein is the carrier of genetic information and led to the coronation of DNA as the molecule...scientists established that DNA encoded all the information necessary for regulating and propagating life...machinery, which can lead to high frequency of indel formation in the genome and are inefficient at introducing...Together, CBE and ABE have obviated the indel formation rates that are typically associated with the DSBs...advertisement of the technology on various non-scientific platforms, CRISPR has also transcended into the world of...

... to avoid contamination, take extra care when performing RNA extraction. RNA is inherently not as stable... protocol with guanidinium thiocyanate-phenol-chloroform. In this method, samples are still homogenized..., water-saturated phenol, sodium acetate, and chloroform are added to the homogenate and shaken. After...ultracentrifugation!), the layers of phenol and chloroform separate, and RNA is retained in the top, aqueous...centrifugation and the supernatant is extracted with chloroform as in the above no-kit method.  Researchers have...Isolation by Acid Guanidinium Thiocyanate–Phenol–Chloroform Extraction. Analytical Biochemistry 162:156–159...isolation by acid guanidinium thiocyanate–phenol–chloroform extraction: twenty-something years on. Nature...

...students first transform the S. cerevisiae met deletion strains and then isolate transformed strains on media...organism into overexpression plasmids. Students next transform deletion strains from the reference organisms ... (7). In our class research project, students transform S. cerevisiae met deletion strains with overexpression...expression of the plasmid-encoded MET gene allows the transformed strain to grow in the absence of methionine (... to select yeast that have been successfully transformed with the plasmids, because the S. cerevisiae ...of MET/Met genes from the plasmids allows the transformed strains to grow in the absence of methionine....

...broken down into four simple steps: Send plasmid information to Addgene Material Transfer Agreement (MTA) ..., we’ve made it easy to send us your plasmid information in a deposit spreadsheet. The data entry process...require that you send your samples to Addgene pre-transformed in this strain. Hazardous If the plasmid...finished filling out the spreadsheet, save it in the format: labname_addgene_batch_upload.xls Replace ‘labname...spreadsheet to [email protected] with the following information: Addgene account username. In order for the ...Yachie, 2021) but can accept sequence files in any format. Distribution status for your plasmids: Hold for...were first created. From there, we’ll use the information in the spreadsheet to generate plasmid pages....

...throughout the conference, what worked? Use this information to prepare for the next meeting. Navigating talks... work to a preprint server (and include this information on the poster) so your visitors can have a DOI...questions from your poster viewers and trade contact information if your conversation is particularly fruitful...’s best to turn to an established social media platform. During the conference If the conference has a...using social media for sharing new scientific information from the conference, many conferences also have...having business cards is a good way to share your information quickly. During the conference Arriving at the...scientific networking Learn how to set up and informational interview and Expand your professional network...

...Your DNA Learn to Do a Bacterial Transformation Learn How to Perform a Diagnostic Restriction Digest ...inverted terminal repeats. We use NEB Stable to transform and prep high quality DNA that contains these ...of your prep. Many plasmid prep kits recommend performing an extra wash step to remove endonucleases (see... in a protein expression strain, we suggest retransforming the plasmid into a strain meant for DNA purification...the binding matrix could clog the matrix when performing filter based plasmid prepping. Be sure to stay...plasmid feature should be indicated in the plasmid information or growth strain instructions from the manufacturer...

... not represent a formal stance taken by Addgene or its staff. Additional information about gene drives...tested in yeast5. Ecological confinement involves performing experiments in a geographic area where escaped...such as malaria and dengue are currently being performed in regions that don't have resident populations...should be used? There are ongoing efforts to develop formal guidelines to answer this question. Until then,...very few gene drive experiments that can't be performed with two or more confinement methods. Since molecular...developed safely and transparently.  For more information, visit www.sculptingevolution.org. References...

...adapted the DETECTR platform to detect SARS-CoV-2 using a lateral flow strip format. They published this... to develop simple, portable, and inexpensive platforms to reliably detect nucleic acids at the atomolar...point, they hope that the protocol serves as a platform for advancing diagnostics.     DETECTR- DNA...harnessed this ability to create a DNA detection platform called DETECTR. How does DETECTR work? DETECTR...technology with the mission to “provide a CRISPR-based platform on which an infinite number of tests” for biosensing...Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6. Science 360:439–...

...builds on our former approach and involves reading primary literature, soliciting information from requestors...requestors or how it performed. Addgene launched our AAV Data Hub as a community platform for the sharing ...upload their experimental data in a standardized format, sharing how well a given viral vector worked —...selecting an AAV can help a researcher make a more informed choice on which AAV to request and/or save time...develop new systems, new partnerships, and new platforms, from our early engagement with UPenn to our launch...

...demographics and the information is often confidential (see image below). Slide of information gathered by Postdoctoral...trying to compile more comprehensive demographic information about the postdoc work force, problems start ...Institutional Perspective. This lack of complete information does not, however, prevent us from identifying...career outcomes for PhDs or postdocs. Having this information publicly available may realign trainee expectations...scientifically minded, and able to communicate complex information - all traits you are likely to find in graduate...Saturday Hack Day Session focused on finding this information and making it more easily digestible/available...

...E. coli can produce your protein. Structural information for many proteins is easily accessible in the...Protein Data Bank and PDBsum. After acquiring this information, you can then pick an expression vector and expression...from the purified protein. For more detailed information on these and other tags used in the pCri system...pathway to improve folding and disulfide bond formation. If one prefers expression in E. coli, the use...-containing vector can improve disulfide bond formation. pCri also includes a special feature for difficult-to-purify... - a database of detailed protein structural information PDBsum - visualization of the data from RSCB ...

...Photoswitches are engineered molecules that induce conformational changes with light stimulation to either activate...receptors. When exposed to light, there is a conformational change in the extracellular domain which activates... OptoXR incorporating rhodopsin undergoes a conformational change when exposed to blue-light and activates...designing your experiment.  Finding the right information When using a previously published system in your...characterization data, like spectral and kinetic activation information, in the supplemental data instead of in the main...have extensive collections of publications and information on hardware, optogenetics tools, viruses, and...important to understand the details of how each tool performs and the consequences of off-target activity. Whether...

... frozen or formalin-fixed, paraffin-embedded tissue sections, methanol- or paraformaldehyde-fixed cell...repeating for each target. Alternatively, you can perform simultaneous detection with a mixture of primary...) for more guidelines for creating clear and informative figures. Pro-tip! If you find yourself stuck ...steps, in order to ensure your antibodies are performing their best and that the sample is not being degraded... P., & Yong, W. H. (2019). An Introduction to Performing Immunofluorescence Staining. In W. H. Yong (Ed...Weissgerber, T. L. (2021). Creating clear and informative image-based figures for scientific publications...

...mind whenever you're working with antibodies.) Conformation changes usually fall into one of two categories...Denaturing  First, let’s think about induced conformation changes. Several types of assays, such as ELISAs..., changing locations, or even just as part of performing a specific function. A transmembrane protein ... validated for the type of assay that you are performing. If they’re not available, the next best thing...denatured protein samples. If, however, you are performing an immunohistochemistry assay, you may want to... immunoimaging assays. It can also elucidate information about what a given protein is doing or changing...

...use. Or you can really capitalize on the dye’s performance and use super-resolution or single-molecule techniques...fluorescent ligand and see the results right away. Or perform pulse-chase experiments by sequentially adding ...-labeled actin filament (488-Actin, cyan), with formin (649-mDia1, magenta) and capping protein (549-CP...priority, SNAP-Tag or HaloTag have been shown to perform differently for different ligands (Wilhelm et al...super-resolution imaging, HaloTag gives better performance than SNAP-tag (Erdmann et al., 2019). Check out...new ligands for these systems, enhancing their performance and ease of use. With so many possibilities from...regulation of actin barbed end assembly by twinfilin, formin and capping protein. Nature Communications, 14(...

...December 2015 we held our first intro to bacterial transformation course (read our blog post on it and to see...consisting of over twenty members and volunteers. Transforming citizens into scientists Anyone can become a...dedicated time at BosLab, he became our bacterial transformation guru and instructor for our intro to biohacking...BosLab. Some are entrepreneurs using our space to perform early-stage experiments. For example, Dave Grawoig...Grawoig is using nanopore sequencing at BosLab to perform proof-of-concept experiments for discovering novel...Addgene and are eager to do our first yeast transformations! Dennis Oleksyuk, Raphael Laurenceau, and Tami...

...force can be generated by conformational changes in proteins? How can information propagate through a structured...monomers respond to pH changes by undergoing local conformational changes. These tiny movements are then amplified...these applications, purified R bodies could also perform as actuators in microelectromechanical systems ...