...phobias–it’s a key part of being a scientist and an incredibly useful transferable skill. Being able to stand...even in my head. What came out, though, was an incredibly calm: “Excuse me, I seem to have lost my place...any silence, to talk. That moment taught me an incredibly valuable lesson: Your presentation will be better...your slide deck gets uploaded. Your mic emits screeching feedback. You’re muted on Zoom. Your video conferencing...provide quick updates during a company meeting increases visibility of your work, opens up collaboration...Conflict Resolution Transferable Skills Guide: Creativity...

...consider scaling up by either increasing the number of 10 cm2 dishes or increasing the vessel size. Opting ...viral genome into additional plasmids thereby increasing the number of recombination events that are required...some cases, titers can drop 10-fold for every 2kb increase in insert size. To overcome this, lentivirus can... lentivirus routinely, you may opt for simply increasing the number of 10 cm2 dishes. While this option...handling multiple dishes will take longer and may increase the risk of mistakes or contamination. Finally...

...2024). It reduces the amount of time to design and create a new vector to four days for an experienced bench...takes a lot of the design work out of the plasmid creation process. Each module of Fragmid represents a class...caption from McGee et al., 2024, used under the Creative Commons CC BY-NC-ND license.   Once you have... system by sequencing 60 individual assemblies created over five months. When they tested those clones... plasmids were perfectly correct. Because they created the assemblies in duplicate, they were able to ...Doench lab share this new tool and hope it helps you create great plasmids for your next CRISPR experiment....

...further modified to create ultrapotent PSEMS (uPSEMs) that have drastically increased potency. Download ...signaling pathways and can lead to such responses as decreased heart rate (Redfern et al., 1999) or altered neuron...firing. Other DREADDs, such as the KORD receptor, increase this threshold and silence neurons. DREADD-activated...actuator modules, were developed with the goal of creating receptors with less endogenous activation and ... Higher potency means lower drug doses, which decreases the risk of off target effects. These synthetic...

...plasmid contains all of the desired gRNAs. This increases the chance that all the edits you want to make... cleave outside of their recognition sequence, creating flanking overhangs. These overhangs can be customized...Expression Systems (MuLE) Kit: This kit can be used to create lentiviral vectors expressing wt humanized Cas9...the gRNAs. gRNAs are flanked by glycine tRNAs to create polycistronic glycine tRNA-gRNA (PTG) constructs...using 3 distinct spacer-repeats, you could easily create 7 unique arrays (e.g. for spacers A, B, and C, ...is possible with efficiency >70%. CRMAGE is an incredibly fast protocol, with only 5 hours incubation time... Find plasmids from this paper at Addgene. 11. Cress, Brady F., et al. “CRISPathBrick: Modular Combinatorial...

...Tsuboi et al.), limiting its use. Nonetheless, its creators noticed a few properties that have now become ... be. Cytotoxicity, improper localization, and decreased functionality are all possible consequences of...al.) Monitor gene expression in the developing pancreas by placing FP timer DsRed-E5 under the control...control of Neurog3 (a gene controlling pancreatic differentiation) in mouse embryos (Miyatsuka et al.) Distinguish...you planning on using an FP timer in a new and creative way? Let us know in the comments section below...differentiates human IPSCs into glucose-sensitive insulin-secreting beta-like cells." Nature communications 7 (2016...

...to, like ones that are hidden in the molecular crevices proteins on the cell surface. Figure 1: ...threonines at positions that can be randomized for creating the libraries (Zimmermann et al., 2018). Find ...protein (sybody), ribosome, and mRNA. During the screening process, displayed sybodies that bind to the biotinylated...the candidate sybodies in their coat protein to screen for binding to the target protein. The protocol...describes going through two rounds of phage display to increase the enrichment of target-specific sybodies. Final...The enriched sybody pools from the phage display screens are subcloned (using FX cloning) into the expression...

... second FP, often decreasing the fluorescent signal from the first FP and increasing the signal from the...an FP to a transmembrane protein significantly increases the effective concentration of the FP. The FP ...to a plane and generally only rotates in 360°, increasing the probability that two FPs will collide with...polymer formation as fusion protein concentration increases. The polymerized fusion proteins that form as ... His interests include the quality control of secretory proteins in the Endoplasmic Reticulum and live...

...well for everyone else?  In truth, people are incredibly varied in how they prefer to consume information...matter will drive the kinds of communications you create. Wildlife biology and ecology can make for beautifully...Once you’ve answered the above questions, you can create your short list of options and then pick whichever...Medium to high Uses visuals, audios, and text. Incredibly immersive and flexibility to visualize and contextualize...format. Equipment, expense and expertise needed can increase exponentially with complexity. Podcasts Medium...audience and communicators. Well-developed content creation platforms.  Algorithms can make it difficult ...

... I’ll describe an episomal vector system that increases reprogramming efficiency through the expression...requires stringent biosafety measures, thereby increasing the costs of the procedure. Episomal reprogramming...reprogramming is an ideal and cost-effective method for creating clinical-grade, safe, nonviral and nonintegrating...episomal reprogramming cocktail by Okita et al. increased the reprogramming efficiency in PBMCs [7]. The...transiently. The transiently expressed EBNA-1 increases protein expression from the other episomal plasmids...used on PBMCs, the pCXLE episomal vector system increases the efficiency of reprogramming from 0.001% to...

...humanities, and 32% in the social sciences go uncited. Creative titles and controversial topics can garner some...Article, since the 1980s researchers have been increasingly citing research from outside of their disciplines...interdisciplinary research while at the same time increasing interdisciplinary citations in their own work...instant sharing across multiple other sites), I created a new platform for sharing video abstracts about...effectively.” How to share your science through video Creating an effective video about your research is not ...offers a 5-step process and several examples for creating effective research videos.  Karen McKee, also...

...cytosine deaminase with Cas9 nickase (nCas9). nCas9 creates a nick in the DNA by cutting only a single strand...inhibitor (UGI) minimizes the second outcome and increases the generation of the desired T-A base pair. ...act on DNA for adenine base editing. They first created a defective chloramphenicol resistance gene (CamR...antibiotic resistance. To find such a protein, they created a mutant library of E. coli tRNA adenosine deaminases...into E. coli harboring the defective CamR gene. Screening for surviving colonies and subsequent rounds of...genotyping. If successful base editing causes the creation or destruction of any restriction enzyme (RE) ...

...Saves Christmas! created by the Moore lab at the Van Andel Institute. Krampus was created by Nate Levine...Warrior The Moore lab, who was full of holiday creativity, also receives an honorable mention for their...2021   2nd Place: The Winter and the Falcon Tube created by the team efforts of the Yit Heng Chooi Lab at... December 4, 2021   3rd Place: O! Beaker Tree Created by Joanne Jary from Lab Support at the John Innes...

...DNA end of the break is resected by nucleases to create a ssDNA 3’ overhang. This will serve as both a ...considerations for designing a repair template to create mutations HDR donors can contain insertable sequences...circular plasmid templates is generally low; to increase the frequency of edits, researchers have designed...complementary ssDNA. Easi-CRISPR was shown to increasing editing efficiency from 1-10% with dsDNA to 25...Frequency of Homology-directed Repair (HDR) for Increased Efficiency of CRISPR/Cas-mediated Transgenesis...

...including easier automation, higher throughput screening, and a lower requirement for the amount of DNA...transgenic animals may choose Southern blotting to screen founder transgenic animals for the inserted transgene...DNA sequence content by comparing the fragments created by different restriction enzyme digests. Incubating...specific fragments of a DNA sequence of interest to create a physical map of the DNA. Starting in the 1970s...below), restriction mapping may still be used to screen multiple colonies before selecting a smaller subset...allowing for fluorometric-based detection. This increased sequencing speed ultimately leading to what are...

...donors to supply surplus lab equipment to carefully-screened research and teaching departments at universities.... Like Seeding Labs, Addgene also feels it’s incredibly important for scientific breakthroughs, reagents...scientific community and are always taking steps to increase plasmid distribution and disseminate knowledge... we need to expand our efforts to find new and creative ways to reach developing countries. As part of... eliminate much of white space on this map and increase plasmid sharing across the globe! We hope that...

... associated hybridoma cell lines. The DSHB was created by the National Institute for Child Health and ... which are each produced by a single B cell. To create a monoclonal antibody cell line, myeloma cells ...individually isolated and expanded. The clonal cells are screened to identify hybridoma lines, each producing a ...and protein tags (c-MYC and HA). We have also created the Monoclonal Antibody Research Institute dedicated...partially humanized rabbit antibodies that exhibit increased avidity to high profile antigens; and 4) generating...

...use the dropdown to increase the length of the list (purple oval in the screenshot below). You can also...plasmid page. Check out the purple arrow in the screenshot below to see what I mean. Additional features...list of papers citing your deposited plasmids Increased visibility of your plasmids in the "Associated...listed in the "Articles Citing This Plasmid" list increasing your work's visibility Help us improve our citations...

.... Figure 1: Diagram of AAV components. Created with BioRender.com. AAV vectors have both distinctive...AAV2, and so on. In addition, researchers have created many engineered serotypes by mixing, matching, ...been shown to transduce skeletal muscle, cardiac, pancreas, smooth muscle, CNS, kidney, and retinal cells...been shown to transduce liver, skeletal muscle, pancreatic, and retinal cells (Issa et al., 2023). AAV9 ...pUCmini-iCAP-AAV9.452sub.LUNG1 Goertsen et al., 2022 Pancreas AAV6, AAV8 Wang et al., 2006 Retina 7M8 Dalkara... J., & Xiao, X. (2006). Widespread and Stable Pancreatic Gene Transfer by Adeno-Associated Virus Vectors...

...specifies the target, the Cas9 endonuclease that creates the DNA double-strand break (DSB) at the target...knock in models). CRISPR mouse model basics To create a mouse model, the gRNA, Cas9, and donor oligonucleotide...selection offered by mouse ESCs. For example, we often screen only 15-25 mice when generating knockout models...for generating mouse models Although CRISPR is incredibly useful for generating mutations by NHEJ and generating...CRISPR, one must beware that not all gRNAs are created equal. Some work better than others. There are ...Mice with Extensive Gene Humanization (25-kbp) Created Using Embryonic Stem Cell/Blastocyst and CRISPR...

...information on black screens, but not to know even the life cycle of the creature they deal with.” So ...Wildlife Magazine. It is easy to see how these incredible images are attracting so much attention. When...efforts, as the subject matter is seen to have increased value and is appreciated by a wider audience for... by holding scientific art competitions where creations can go on display for all to enjoy. The largest...

... Bauer's labs details the use of CRISPR/Cas9 to create genomic deletions in mammalian cell lines. Below... paired DSBs. This method allows for deletion screening by conventional PCR. Biallelically disrupted cells...available strategies by which biologists can imagine, create, and examine novel genetic configurations. Watch...transfected cells via FACS, primer validation, and screening by PCR for biallelic deletion clones:    ... and delivery, as well as their techniques for creating deletions, identifying these deletions, and finally...

... your research! This time: GeneWeld vectors to create knock-ins CasX Drug inducible CRISPR/Cas9 activation...CRISPR knockout libraries for cancer research Creating knock-ins with GeneWeld vectors GeneWeld from ...Essner’s lab uses CRISPR/Cas9 and a donor vector to create knock-ins of fluorescent proteins, fusion proteins...library with SpdCas9 to perform pooled CRISPRi screens to identify essential genes and phage host factors...metastasis cohorts and a prior mouse metastasis CRISPR screen. The Chen Mouse Tumor Suppressor Gene CRISPR Knockout...

...reliably detect single-base differences. The original creators of this technique visualized the ligation product...quencher pairs or qPCR-based methods to greatly increase the sensitivity of detection (14-20). LDR has ... 27). This methodology has been used to rapidly screen buffer conditions for Taq DNA Ligase and their ...electrophoresis (CE) (see below). This method allowed screening of all possible base combinations (Watson-Crick...of a 96-well plate, allowing 6 conditions to be screened per plate. The results indicated that the optimal...optimization method has been used internally at NEB to screen additional ligases, conditions, and formulations...

...lentiviral vector system. Viral vectors have been increasingly popular in fundamental and applied research ...replicate. However, Mother Nature found a way to re-create a functional virus. Indeed, by testing several ...the packaging plasmid forming a vector that could create replication competent virus. Even though this system...non-dividing cells and so researchers set out to create a better, safer system. Unlike the MoMLV, lentiviruses...vectors have been established so far, with safety increasing in each generation. The 1st generation of lentiviral...protocols suggest adding sucrose to the supernatant to create a gradient of density that favours virus precipitation...

... MOIs. 2. Create a monoclonal Cas9-expression cell line. Especially if you’re doing a screen. I made a...who did not tell you that essential thing ask incredulously, “You seriously didn’t add 3 microliters of ...is bad news for repeatability and bad news for a screen. Because Addgene viruses are provided at a specific... vectors and genome-wide libraries for CRISPR screening. Sanjana NE, Shalem O, Zhang F. Nat Methods. 2014...Additional Resources on the Addgene Blog Genome-wide Screening Using CRISPR/Cas9 Deliver Multiple gRNAs at Once...

... strategically to increase your rate of productive results and thereby help decrease the time to your ... to achieve your intended next career stage and create a timeline for achieving these goals. Planning ...ways to consolidate or present your data. You can create more detailed or functional models or more helpful...mentor, manage, communicate, and teach The dirty secret about becoming a group leader is that people are... His interests include the quality control of secretory proteins in the Endoplasmic Reticulum, optimization...

...activities (1). She has developed a fast endophyte screening system and plans to employ the light‐inducible...expert. In order to preserve Cassava yields and to increase the potential for this drought tolerant plant,...significant proteins. In this project she aims to create transgenic yeast as an alternative source of essential...plasmids. The recombinant Cry proteins will then be screened for toxicity against local insect pests. By developing... use of chemical pesticides may be avoided or decreased. Biosensors for herbicide detection Dr. Rodrigo...

...CRISPR technology has brought drug and genetic screens together and expedited the drug discovery pipeline...Alternatively, a disease model can be generated, and drugs screened for a change in pathology. If the disease pathology...accelerate the drug pipeline. High-throughput CRISPR screens can be used to inhibit, activate, or knockout many...or an entire pathway of possibilities greatly increases the odds of finding a quality drug downstream....identified several candidate inhibitors of gene X in a screen. You generate a gene X knockout in a cancerous ...

...isolated and screened. Depending on the marker, traps can be used as high-throughput screens to introduce...models. Why traps are useful Traditional genetic screens employ various on/off switches to measure the function...circumstances, trap methods may be the preferred screening option. Promoter traps Promoter trap systems ...101: Using Transposons in the Lab Plasmids 101: Screens vs. Selections Plasmids 101: Knockout /Knock-In...

...CRISPR screens symposia outlined the fantastic work this Broad Institute lab is doing creating well-optimized...well-optimized CRISPR screening technology. Addgene got several shout-outs for making popular screening libraries ...presence in the microbiology community. For our first concrete steps into this field, we’ve curated microbiology...

...pENN.AAV.hSyn.HI.eGFP-Cre.WPRE.SV40 AAV2 Recombinase James M. Wilson New serotype pENN.AAV.hSyn.Cre.WPRE.hGH AAV2 Recombinase....GFP-Cre.WPRE.SV40 AAVrg Recombinase James M. Wilson New serotype pENN.AAV.CamKII 0.4.Cre.SV40 AAVrg... viral prep AiP14825: pAAV-AiE0441h_3xC2-minBG-iCre(R297T)-BGHpA (Alias: CN4825) AAV-PHPeB Recombinase...

...cobalamin fusion to the riboswitch results in increased fluorescent signal. Prior to the development of...developing a new imaging-based bacterial colony screening platform, Oliver Griesbeck’s lab set out to engineer...the red end of the spectrum (above 600 nm) and increasing the brightness. Arne Fabritius and colleagues...Listen to the base editing podcast segment The increasing popularity of single base-editors has resulted...cytidine deaminases like APOBEC. To achieve this they created a lentiviral vector system that monitors both APOBEC...

...large-scale functional screen in bacteria. Sensor variants were then refined by a secondary screen in rat hippocampal...protein (CREB) in living cells. “Now we are making this really good,” Griesbeck said. “We know CREB is really...10.1038/nmeth.2773. (PubMed) Friedrich et al. Imaging CREB activation in living cells. J Biol Chem. 2010 Jul...

...photoconverts predominantly to the anionic form, causing increased fluorescence when excited with 488nm blue light...which allows PA-GFP to exhibit up to a 100-fold increase in fluorescence when illuminated with blue light...performing several rounds of random mutagenesis screening for enhanced RFP variants. PA-mCherry (E26V / ...photoswitching. In 2008, scientists used mutagenesis screens to find a version of Dronpa that doesn't become...the absence of photoactivation resulting in an increased signal to noise ratio. PA-FPs with higher contrast...super resolution microscopy, PA-FPs have helped create several new imaging methods, including photoactivation-localization...

...roughly 200 cells. Increase the titer to 1013 resulted in a roughly 6-fold increase in expression [3, ...Figure 2]. Note how expression increases exponentially with an increase in titer. That means that once...to get more AAV in contact with the cell is to increase the AAV concentration. In this flowchart, you ...

...primers to create 200-300 bp sequencing products using PCR. A diagram of the product we created for the ...plasmid. However, lots of library DNA is needed for screening experiments, so the scale of the DNA prep is larger...rank the plasmids by abundance, scaled to 1, to create a “cumulative wealth distribution” curve, or Lorenz...wishes you luck in your future library use and screening, and if you have any tips, input, or stories of...

...addition to increased brightness, they also found that the signal to noise ratio can be increased from 2-fold...previous CRISPR-based loci visualization tools by increasing gRNA stability. First, they began with the Broccoli...and went through subsequent design iterations to increase stability and minimize misfolding and recombination...label two loci simultaneously, Ma and Pederson created another sgRNA containing the PP7 hairpin sequence...

...protein secretion, making harvesting recombinant proteins a cinch. This is because it doesn’t secrete its ...of producing high protein animal feed. Due to increasing prices of methanol and advancements in other ...proteins, making the recombinant protein its major secretion. Beyond sheer protein production, K. phaffii is...Organisms Plasmids 101: Yeast Vectors Tips for Screening with the Yeast Two Hybrid System Additional...

... lack of credit for small contributions. We strive to eliminate these two barriers, creating a community...with others than by oneself that prompted us to create Crowdfight.   Figure 1: Crowdfight is a ...collaboration between researcher and expert; 4) Credit all contributions.   Our volunteers are extremely...collaborations can be easily started and easily credited. And nothing stops these one-time favors from ...