...Every few months we highlight a subset of the new plasmids and viral preps in the repository through ...CRISPR corner By Jennifer Tsang Here are a few highlights from recent CRISPR plasmids. To find all of the...Plasmids and Resources page.  A new system uses light to deactivate photocleavable gRNAs within seconds... assembly of CRISPR/Cas9 constructs. zCas9i is a high efficiency intron-optimized SpCas9-coding gene. ...

...very powerful for high level recombinant protein expression. These systems enable high expression of very...biology research (eg. polymerases and other enzymes might be purified and used to manipulate DNA), or in medicine... through stable cell lines. Both methods produce high protein yields if transfection is successful. Some...if a desired protein product is toxic to cells at high concentrations. Despite their advantages, mammalian... promoter and induced by lactose, provides the highest level of protein expression. Despite their ease...

... trying to perform high-throughput experiments. If a user wants to carry out a high-throughput genetic...the DSB. CRISPR has allowed scientists to tag and light up endogenous genes of interest to better understand...convenient one tube assembly reaction to generate high-throughput targeting vectors. These vectors can ...fluorescently-tagged proteins at endogenous loci, shedding light on previously unknown protein functions. References.... and Eric M. Jorgensen. “SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis...

... use to introduce yourself and what you do. You might want to include your university and your advisor...new at a conference, one of the first things you might want to know is what their project is. Or maybe ...familiar with. You can tweak your elevator pitch slightly if you know a bit more about the other person....elevator pitch several times at the conference, you might notice some trends. Were there parts of your elevator... up on the board and meet your poster session neighbors. During the session, if someone comes up to my...and your resume and prepare business cards. You might not consider being a student or postdoc a “business...

...Every few months, we highlight some of the new plasmids, antibodies, and viral preps in the repository...nucleocapsid RNA chaperone, RT: reverse transcriptase. Right: T1 tomato plants that inherited an edited gene ...rely on synthetic dye molecules, which are much brighter and more photostable than fluorescent proteins...Craggs, T.D., & Twelvetrees A.E. (2024) Kinesin-1 is highly flexible and adopts an open conformation in the...illumination regimes with fast activation kinetics and high responsivity near the resting membrane potential...

.../s12915-019-0682-0. Beck, C., Gong, Y. A high-speed, bright, red fluorescent voltage sensor to detect...the kinetics of voltage sensing to signal output slightly slower than other approaches. Functionally, the...photobleaching, lack of red-shifted indicators, and brightness of fluorescence all have room for improvement...transport, not fluorescence, they are not nearly as bright as the other GEVI systems that use engineered FPs...one component counterpart and is significantly brighter and available with red-shifted fluorophores (find...

...Association for Cancer Research Annual Meeting, you might consider including more background on how your virus...to expertly show off your science.  Prepare the night before: To avoid rushing out the door without your... tack up next to your poster. Arrive early: You might be nervous before your poster session, or it may...walk up to someone’s poster, and then he/she dives right into the background without an introduction. If ... in his/her work, and would enjoy hearing the highlights so that you can appreciate the impact of the ...

...Kits can also have the annoying tendency to runout right when you need them and to accumulate a bunch of ...precipitated, dried, and then rehydrated. There are some slight modifications depending on the type of DNA you’... not bind silica or glass, but the addition of a high concentration of a chaotropic salt (guanidine hydrochloride...: agarose gel slices Product: DNA fragments You might be wondering, “How is milk going to help me extract...purify DNA ranging in size from 100 bp to 48 kb with high yields and without degradation during the extraction...nucleic acids. Containinants bind cellulose less tightly than nucleic acids, so placing the dipstick into...

...). I’ve assembled a series of screenshots to highlight changes to both the design and functionality of...involves delivering a calcium biosensor using AAV, you might decide you’d really prefer to order ready-to-use...look at filters in a moment. First, I want to highlight the convenient ways our new search tool lets you...you know the ID number for a plasmid, your straightforward search is now even easier with a direct link...

...average, AAV.PHP.eB and AAV.PHP.S had 1.5- to 2-fold higher GFP intensity compared to the strains they were...were derived from. They also transduced a higher number of neurons overall (GFP+ cells that also stained...dendrites extend to. The keys to this method are 1) high levels of color diversity so that cells are labeled..., or blue (RGB) fluorescent protein (4a-c). When high doses of this viral cocktail are injected, by chance.... tet-off transactivator (tTA), is injected at a high dose to maximize the number of cells that are labeled...part system, color diversity is maintained at both high (figure 4e) and low doses (figure 4f) of the tTA...

...making the purification process simpler. Due to its high yields, E. coli is often used for the production...transfection, depending on whether the heavy and light chain genes are contained to one plasmid or if they...on an SDS-PAGE gel. Once stained, the heavy and light chains become visible alongside your ladder and ...software. Any bands on the gel beside the heavy and light chains can also be measured. Contaminating bands...Rockberg J, Chotteau V. Small-scale bioreactor supports high density HEK293 cell perfusion culture for the production...capture process based on magnetic beads from very high cell density suspension. Biotechnol Bioeng. 2021...

... information about insert orientation, then you might consider designing orientation-specific primers....primers. Blunt end cloning is an example of when you might want to know the orientation of the insert. One member...with this and a few other colony PCR tips are highlighted below. Preparing template: Pick a single colony... liquid cultures. Lastly, you can start small overnight liquid cultures with the clones you pick and only...size. Make sure to use an appropriate molecular weight standard for reference and to add a loading dye...

...James from Binning Singletons. During Junior High and High school in the 80s and 90s, Joe was that kid ...asked if she had a few minutes to point me in the right direction for my future work. She graciously took...match people within similar fields (ASM Microbe has eight tracks), something we didn’t have enough people ... ASM Microbe, we connected people who otherwise might never have met. Binningsingletons.com will be the... but before going to graduate school, Joe taught high school chemistry for a year. The teaching/mentoring...

... advice on areas where you might need some help. Today, we’re highlighting an oft-underlooked transferable...skate as a starting point, their ideas scored 37% higher in originality. It may not be easy to optimize ...truly disagrees with the group (because they will fight more adamantly for their solution), selecting a ...power to change anything. You may not have the highest position or the longest tenure, but don’t let that...

... confirmed that indeed the gRNA-pMB1 had a slightly higher leaky expression in BL21(DE3) that resulted...customizable cargo gene in the form of RE-cargo-LE (RE, right end; LE, left end) pTnsABC expresses the transposases...glucose dehydrogenase expression that is 2.6 times higher when compared to the industrial strain. In our ...copies of integrated genes. We obtained a stable and high-yielding strain which had 6 times the yield of the...transfer on transposition-inducing agar lead to higher cargo copy numbers. (D) The effect of gene dosage...

...Genome-wide CRISPR/Cas9 screens are a high-throughput systematic approach for identifying genes involved...alternative to genome-wide RNAi screens, which although highly effective, are affected by low on-target efficacy...unclear if off-target cuts confound the results of high-throughput CRISPR screens. Do short guides (17-...mismatches more distal to the PAM, and 2) guides with high GC content have greater off-target activity. Short... with only a small loss of on-target activity in high-throughput screens. The human and mouse versions...measurement of off-target activity using Cas9 toxicity in high-throughput screens." Nature communications 8 (2017...

...our plasmid lab to develop a reliable and robust high quality viral vector service, making an array of...protocols we use in our lab — being able to order high quality viral vectors from the Addgene repository...on quickly in the scientific community, turning slightly under ten requests a day in 2017 to over fifty...that are activated, respectively, by substrates, light, or engineered ligands (Figure 2). Biosensors, which...our own repository data, giving us significant insight into which vectors are likely to be of most use...plasmid repository gave us the experience to launch a high-quality ready-to-use viral vector service. But in...

...chance of cross-reactivity Sandwich High specificityHigh sensitivity Longer protocolIncreased chance...several drawbacks. Direct ELISAs generally have higher levels of background staining. Antigen-containing...simple protocolReduced chance of cross-reactivity Higher level of background stainingLess sensitivityLess...availability of conjugated antibodies Indirect High sensitivityGood antibody availability Longer protocolLess...Antibodies 101: ELISAsAntibodies 101: Selecting the Right Antibody Antibodies 101: Polyclonal Antibodies Resources...

...which cuts at NRN PAMs with high efficiency, as well as NYN, but with a slightly lower efficiency. Information...available, it can be difficult to decipher which one is right for your unique experiment. Here, we will introduce...the genome and consider using a Cas9 variant with higher specificity (eSpCas9 and HypaCas9) instead.  Choosing...location and type of edit you need to introduce highly specific? Perhaps you need to introduce a single...choose from within the Cas family if Cas9 isn’t right for your experiment. Keep an eye out for our upcoming...

...up-regulated. For instance, generally, the more highly expressed a gene is under native conditions, the... achieve using a CRISPRa. Your gene of interest might already be hitting an upper bound of activation ...scale CRISPR-based screens can be used to gain insight into protein structure and function (Pan et al...., Weiss R, Aach J, Collins JJ, Church GM (2015) Highly efficient Cas9-mediated transcriptional programming...doi.org/10.1016/j.cell.2014.09.029 Konermann S, Brigham MD, Trevino AE, Joung J, Abudayyeh OO, Barcena ...j.cels.2018.04.011 Polstein LR, Gersbach CA (2015) A light-inducible CRISPR-Cas9 system for control of endogenous...