...the least expensive of all the options discussed as it only requires PCR and the inexpensive DpnI enzyme...here to help with that! Four important factors to consider for any experiment are: Speed Cost Difficulty...For cloning in particular, another important consideration is: The number of inserts ...as some methods...the following cloning methodologies have been extensively described in other articles; so if you are interested...little as 30 minutes! Alternatively, you might consider Gibson cloning. In this technique, you first perform...order determined by their homology. You might also consider FastCloning, first described by Chaokun Li and...Finally, TOPO cloning is another good option to consider when time is of the essence. This method requires...

...cellular-level events and behavior. It’s a problem that he considers “one of the greatest challenges of neuroscience...particular advantage for observing events taking place inside cells that are on the move, such as T lymphocytes... calcium-binding sites as possible for added sensitivity. As reported in Nature Methods, the FRET changes...essentially all of the standard organisms for which extensive genetic tools are available. While he recognizes... he said. “It gives you more information than intensity-based readouts.” Griesbeck also points out that...approach he and his colleagues took to optimize sensitivities can now be applied to dozens of other published...for visualizing the transcription factor cAMP-responsive element-binding protein (CREB) in living cells...

...classes of optogenetic tools, and what factors to consider when choosing one.     Figure 1: Example...or inhibit specific neurons without needing to consider secondary signaling pathways. There are many different...different opsin (natural and engineered proteins responsive to light in combination with retinal) variations...reviewed in Abreu & Levitz, 2020), including light-sensitive chimeric GPCRS (OptoXRs) (Tichy et al., 2019),... the extracellular domain of endogenous light-sensitive receptors, like mammalian rhodopsin, and the intracellular...Created with BioRender.com.   Important considerations for using optogenetic tools Once you’ve selected...specific tool. There are many, many options to consider, and the following factors will help you determine...

... One consists of NLuc fused to enhanced green fluorescent protein (GpNluc) and the other consists of NLuc... (FRET) probes. These probes generally consist of a responsive protein (e.g. calcium binding protein) ...membrane-inserted voltage sensing domain (LOTUS V; Luminescent Optical Tool for Universal Sensing of Voltage). With...how bright you can make a reaction but for how sensitive you can make a reaction. NLuc requires 100-fold...) fused to two fluorescent proteins. When the responsive protein binds to calcium it changes conformation.... In well-designed FRET-based biosensors, the intensity of the FRET signal can be correlated to specific...No photoexcitation High Ion specificity High sensitivity Easy expression Gives ratiometric data The result...

...your plasmid yield is sub-optimal? If you are consistently getting sub-optimal plasmid yields from your...Addgene uses and recommends DH5𝝰! This strain consistently gives good, high quality preps due to its endA1... this handy guide from OpenWetWare for a more extensive listing of common E. coli strains. Increase the...media or adding supplements can improve your cell density, leading to an improved plasmid DNA yield. Culture...supplements to the media You can achieve higher cell density using a nutrient-rich broth such as Terrific Broth...in moderation) may also serve to increase cell density and plasmid yield. Start with a single, fresh colony...Increasing the incubation time can improve the cell density and therefore the plasmid yield. You can optimize...

...each event to a channel number based on intensity. Higher intensity events are assigned to higher channels...a user will compare the shifting intensity of events. Since intensity positively correlates with expression...cabinet with cloning rings or following labor-intensive limiting dilution protocols? If this sounds like... the cell’s size, complexity and fluorescence intensity.   What flow cytometers detect Cell size and... a high degree of internal complexity such as extensive membranes produces a greater SSC.  Protein expression...The electrical system converts the fluorescence intensity to a voltage pulse, called an event, and assigns...reporter on the N- or C-terminus of the protein. One downside of fusion proteins is that the tag can alter the...

... to be considered as well. In this post, we’ll touch on antibody storage, buffer considerations, and give...records, and consider the following questions as you move forward:  Was a light-sensitive conjugate on...you’re planning to run with the antibodies time-sensitive, using precious samples, or being compared to ...data in a manner that demands high accuracy and consistency?  If the answer to any of these questions is...saving the poor, abandoned antibody for a…less sensitive moment.   Fig 1: Blugene fading into darkness...fix it. HRP is easy to find, simple to use, not sensitive to light in its inactive state, and cheap. Unless... panel in the machine. Fluorophores are light sensitive, so antibodies conjugated to them need to be stored...

...more sensitive channelrhodopsin actuator for triggering action potentials at low light intensities, the...cumbersome. To overcome many of the limitations intrinsic to DNA-guided systems such as TAL effectors or...antibodies fused to sfGFP. This system amplifies the intensity of fluorescence signal and enables tracking of... H+ specific response cytoplasmic retention insensitivity to other ions In BRET, A mutant Renilla luciferase...dimers by fusing the SsrA peptide with the light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2...activator protein to drive transcription. The anchor consists of dCas9 fused to CIB1 and is targeted to specific... a photoswitchable fluorescent protein which transitions from emitting orange (λ= 565nm) to far-red (λ...

...fluorescence intensity remains. The first issue is that, in several cases, the fluorescence intensity decay ..., fluorescent proteins may display reversible intensity changes (Shaner et al, 2008; Bindels et al, 2017...experiment is performed that measures fluorescence intensity over time. In order to predict how fluorophores...proteins and by comparing how the fluorescence intensity changes over time, the photostability of the fluorescent...photobleaching rate does not depend on fluorescence intensity or protein distribution. Therefore, photostability...analogous to radioactive decay). Consequently, the intensity decay is often described by the t½, which is the... it is necessary to know how the fluorescence intensity evolves over time. The second issue is that, in...

...help with transitioning out of academia (because so many scientists are making this transition). Another...have experience and knowledge to help scientists transition out of the academic sphere into a different meaningful...not always as effective in mentoring for this transition. Second, a coach will help you set goals and ...successful outcome and the good ones take this responsibility seriously. As I make my rounds speaking at ...However most coaches who focus on scientists are sensitive to this and have coaching plans that are financially...here are a number I have come across for you to consider. Feel free to add other suggestions in the comments...scientific backgrounds. For a stated purpose of transitioning a scientist from academia to industry, you might...

...2024.12.20.629623.  A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic events.... Z. (2024). A fast and responsive voltage indicator with enhanced sensitivity for unitary synaptic events... tags for single-molecule biophysics Fast and responsive voltage indicator ONE-GO biosensors kit Antibodies...collaborators recently created ASAP5, a faster and more responsive genetically-encoded voltage indicator (GEVI) ...ASAP5 selected for its improved kinetics and responsivity in screening ASAP3 variants. B) Electrical and...regimes with fast activation kinetics and high responsivity near the resting membrane potential, ASAP5 is...the formation of Gα-GTP by BRET. The biosensor consists of a YFP-tagged G protein and a NanoLuc® (Nluc...

... problem. It is estimated that mycoplasma is responsible for up to 60% of the cell culture contamination...contamination (Uphoff, 2002). Mycoplasmas are considered to be one of the simplest and smallest bacteria. The absence... source of culture is irreplaceable or highly expensive). Detecting mycoplasma contamination Detection...Drexler, 2011). Culturing on agar plates/broth is considered the ‘gold standard’ for detecting mycoplasmas...noticeable color change. This method has high sensitivity but low specificity (Degeling, 2012). Bioluminescence...result interpretation might be difficult. The low density of mycoplasmas, contamination by other bacteria...you keep all the required equipment and reagents inside the laminar airflow before and while you are working...

...Gervais C, Grazzini E, Durocher Y. Transient Gene Expression in Suspension HEK293-EBNA1 Cells. Methods Mol...based on magnetic beads from very high cell density suspension. Biotechnol Bioeng. 2021 Sep;118(9):3499-...At its core, harvesting recombinant antibodies consists of separating antibodies from media, cells, and...this method, beads are incubated with the cell suspension. Antibodies bind ligands on the beads and a magnet...alongside your ladder and a protein standard. The density of the antibody bands can be measured against the...multiple contaminating bands or a few strong ones, consider increasing the number of washes performed during...conformational changes in the antigen or lower detection sensitivity of the assay. Where possible, it can be useful...

...system both favors antibiotic sensitive bacteria and prevents sensitive bacteria from acquiring resistance...system. These CRISPR antimicrobials successfully resensitized a beta-lactam resistant population to the antibiotic... may result in plasmid loss and antibiotic resensitization, or may also cause cell death. Image from Beisel...et al. designed a two-phage CRISPR system to resensitize antibiotic-resistant bacteria (Figure 2). The...Bacteria that receive this phage are now antibiotic sensitive, but lytic phage-resistant. When the lytic phage...killed, antibiotics can be used to target the sensitive population. Yosef et al.’s proof-of-concept work....   Figure 2: Two-phage strategies to resensitize bacteria to antibiotics. A lysogenic phage infects...

...also incorporated LOV-Turbo into TransitID experiments.  Find TransitID plasmids here! These two developments...genetic cargo Optogenetic proximity labeling and TransitID Tracking cellular history with protein ticker ...37612276.   Optogenetic proximity labeling and TransitID By Mike Lacy Proximity labeling can identify protein...utility of this technique.  By engineering a light-sensitive LOV domain into the TurboID enzyme, Lee and Cheah...plasmids here! Next, Qin and Cheah et al. described TransitID, where two proximity labeling enzymes, TurboID...catalyzing the biotinylation of nearby proteins. B) TransitID uses sequential labeling by TurboID and APEX2 ...trafficking within and between living cells by TransitID. Cell, 186(15), 3307–3324.e30. doi: 10.1016/j....

...Cell lines should be routinely subcultured at densities that are low enough to prevent confluency but ... in lower lentiviral titers. Another factor to consider is the age of your culture post thaw. In general...can be subtle and may include reduced saturation density, decreased rates of growth, or clumping. To ensure... and resource consumption due to the high cell density of the culture. In addition to benefitting your...lower titer virus. In such cases you may want to consider concentrating your viral prep. Storage can also... start to a successful infection, one point to consider is how the virus is being titered. Most titering... that routinely produce lentivirus may want to consider testing a variety of cation and serum sources ...

...in yeast with Rosella, a pH-sensitive fluorescent biosensor A pH-sensitive fluorescent biosensor-based...-emission biosensor Rosella consists of DsRed connected to a pH-sensitive variant of GFP (SEP) by a 9 ...Virus (WNV) induced cell death. The Wu lab library consists of 77,406 individual gRNAs targeting a total of...Moduclar Cloning (CIDAR MoClo) Parts Kit. This Kit consists of a variety of plasmids containing many different...plasmids to the scientific community. This collection consists of plasmids containing genes from a crowd-sourced...scientists use GMAP, there will be a continuous expansion of compatible backbones, promoters, and genes ...all under the control of the cI857 temperature-sensitive repressor. The temperature-controlled expression...

...experiments a bit more challenging. With these considerations in mind, when planning an experiment users ...transgene, and downstream applications should all be considered when choosing the correct system. Recently, ...volume. Consequently, you will end up with a considerably lower volume than with the unconcentrated prep...experiment requires a certain volume of virus, consider scaling up by either increasing the number of ...while the PEG precipitation method is relatively inexpensive and does not require an ultracentrifuge, PEG ...lentivirus, it is time to titer. One important consideration when titering a lentivirus is the cell line ...transducing a target cell line. One should also consider the specifics of the titering transduction experiment...

... example, while gene knockouts have long been considered the “gold-standard” for LOF studies, the high...complementary to the dsRNA (Wilson and Doudna, 2013). RNAi consists of two non-exclusive gene silencing pathways: ...data generated from both methods did a more comprehensive gene list emerge that accurately reflected the...Consequently, I will focus on different approaches and considerations present at the first stage of an RNAi-based... of the RNAi trigger requires a few initial considerations, such as the starting material (DNA-based vector...target sequences are concatenated. hpRNA has been extensively used in plant research and many different constructs...further rounds of RNAi. This process, known as “transitivity,” calls into question any claims about specificity...

...the tool, and develop new applications for magnetoresponsive proteins. Motivated by curiosity Although...Maria dreamed about the possibilities of a magnetoresponsive fluorescent protein. Controlling an optogenetic...Frank Hayward et al., 2024). The fluorescence intensity decreased whenever the magnet approached the sample... in 2023.)     Figure 1: Fluorescence intensity of EGFP-FlavinTag in E. coli changes in response...self-labeling FlavinTag made a reliable one-component magnetoresponsive system that could be expressed and purified...cofactors.      Figure 2: Fluorescence intensity and ΔF/F of MagLOV, a variant of AsLOV2 with mutations...with other engineering approaches and that magnetosensitive tools can be developed from other fluorescent...