... 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...

...specific system (by measuring the fluorescence intensity); and 2) to visualize the localization of the ...thus tracking the localization of that biomolecule inside living cells. FPs classified by the emission color...range, there are other traits that need to be considered when choosing an FP: Unique categories of fluorescent...distance between the two FPs is <10nm, and should be considered when labeling proteins that interact. Indeed,...Temperature: FPs maturation times and fluorescent intensity can be affected by the temperature. For instance...experimental parameters (e.g. excitation light intensity, pH or temperature). Photostability: Fluorescent...experimental parameters (e.g. excitation light intensity, pH, or temperature). pH Stability: This parameter...

...biologists were no longer beholden to chemists and (expensive) synthetic fluorophores. Add a dash of DNA with...to small-molecules: red-shifted spectra, ion sensitivity, photoactivation, etc. These impressive advances...requires more photons. For example, moving from transient overexpression of protein fusions to gene-edited... HaloTag, TMP-tag) – These widely used systems consist of a genetically encoded enzyme variant tag that...fluorogens (Figure 1e). Stains – These labels consist of a fluorophores conjugated to molecular species...can be made fluorogenic using environmentally sensitive fluorophores. Overall, self-labeling tag systems...groups (Figure 2b), which can preclude their use inside living cells. We recently discovered that incorporation...

...tissue needs to be considered. Q2: Can I use lentiviral transfer vectors for transient transfections? A2...dividing and non-dividing cells.  Addgene has an extensive collection of lentiviral plasmids created for ...plasmid backbone that was designed specifically for transient expression (find some useful backbones here) or... or from viral transduction. While not ideal, transient expression of viral constructs is a useful tool...cell line. When expressing a viral construct transiently care needs to be taken to ensure that the proper...differ in two ways. Firstly, 2nd generation systems consist of an envelope plasmid and a packaging plasmid ... for the HIV Tat protein and consequently are considered safer. Second-generation transfer plasmids MUST...

...develops an enhanced propensity for movement and activity called locomotor sensitization. This enhanced movement... modulating. Viral vectors, in contrast, are considerably more versatile and specific in how they manipulate...ve previously discussed some of the general considerations for performing behavioral tests, but now we...seeking drugs’).  What’s involved in locomotor sensitization? Previous research has shown that regions such... glutamate transmission leads to locomotion sensitization. (for review see van Huijstee & Mansvelder  ...suggested inhibitory mechanism of locomotor sensitization and might open new ways for a novel therapeutic...subset of excitatory neurons (Drd1) in mPFC is responsible for the antidepressant effect. To specifically...

...come up with a list of things to consider as you make this transition. Of course, a “desk job” could entail...employee who's coming directly from grad school. The transition has been a bit rough for her (3 weeks in and ...there are any resources that can help with the transition.” The lab-to-desk-job culture shock is a real...scientist colleagues have as well. Making the transition from the bench can be jarring for the unprepared...resources out there for scientists making this transition. In this installment of the Addgene career guide...What should you expect during the lab → office transition? I’ve already provided a few counterpoints to...likely happen eventually. If you approach that transition dreading the change, the adjustment period will...

...training schedule that helped me transition easily. Training and responsibilities Through this training, I was...The application process When I first saw the comprehensive job description for this co-op, I was immediately...me a full description of the tasks I would be responsible for, in comparison to the other positions listed...for the independent work on the QC team. I was responsible for helping other scientists on the team ensure...sequencing QC work    In addition to my responsibilities on the QC team, I was able to explore other...uses something called “core hours”. Core hours consist of the time between 10 AM and 3 PM, when most meetings... the “9-5” was not as scary as it seemed. The transition into a traditional work environment was aided...

...Optogenetics, the use of light sensitive proteins (opsins) to manipulate cell activity, enables researchers... with fluorescent tags and activation of light sensing proteins. Furthermore, during the first 2 weeks...Optogenetics functions through opsins which are light sensitive proteins. Opsins used in optogenetic studies are...these charged particles and whether they make the inside of the neuron more positive or more negative.  ...  Neuronal stimulation: Depolarization When the inside of the neuron becomes positive over a certain threshold...inhibition: Hyperpolarization Conversely, when the inside of the neuron becomes more negative it becomes ...differing peak absorption wavelengths and light intensity requirements of different opsins, studies have...

... being considered for the analysis. (C) A more liberal gate allows more cells to be considered for the... SSC values depend on the structural complexity inside the cell or on its surface. Bear in mind, the voltage...histogram, which graphically represents the time and intensity of the cell’s illumination (see Figure 2). H describes...potential that can be applied to the photomultiplier inside the cytometer to increase the electric current ...as our cells of interest do not lie beyond the dimensions displayed in the plot. (B) A more restrictive... marker stains of interest). Let’s double-click inside our gate and proceed to the next plot! Pro tip!... on to my experimentally-specific gates. Two-dimensional staining (quadrant gating) For our last example...

...A major limitation of APEX was its low sensitivity. Sensitivity could be increased with higher expression... avidin, proximity labelling can capture weak/transient protein interactions as well as be used for both...promiscuous biotin ligase (Kim et al 2016). BioID2 consists of a humanized biotin ligase from A. aeolicus,...cell.   Find BioID2 plasmids here APEX2: A more sensitive ascorbate peroxidase for proximity labeling A ...aggregation. The Ting lab hypothesized that the low sensitivity was due to suboptimal folding or stability. Thus...identified a mutant with significantly increased sensitivity that did not affect the function or efficiency... assemble when in close proximity. Split-BioID consists of a split BirA* molecule that can reassemble ...

... same considerations apply as for standard IF. However, there are a few specific considerations to keep...standard immunofluorescence, but with some extra considerations (Im et al., 2019; McLaughlin, 2019).    ...a good place to get started.  One important consideration when deciding whether you can do simultaneous...suitable antibody against your target protein, consider fusing a protein tag to the target and using an...epitope-based strategy such as Anti-6xHis. You could also consider strategies using other types of affinity reagents...your experiment, you might need to quantify the intensity of the different signals in a certain region or...optimizing your blocking and washing conditions or consider using a different antibody.  Prepare a positive...

... are considering a career at a small, teaching college you might want to consider less expensive, portable...low salary I might add). You have a right and responsibility to choose a lab where you can thrive and do...Former lab members can give you a fuller picture. Consider talking to people in the lab across the hall or... hope to grow.  Find mentors who take their responsibility to train you seriously. Why work for someone...whether or not a lab head is a good role model, consider questions like: How long do lab members stay ...allocation of meaningless tasks, removal of responsibility, repeated reminders of blunders, setting target...equipment quotes? Understand administrative responsibilities? Is the work (topic and/or technical aspects...

...variation between iPSC clones. Retroviruses are not considered safe for clinical applications.     Lentiviral...Like retroviruses, lentiviral delivery is not considered safe for clinical applications. Excisable Cre-lox...same as using a standard lentiviral vector, but transient expression of Cre recombinase allows for deletion... site of excision, this approach is still not considered safe for therapeutic applications. piggyBac (...insertional mutagenesis, adenoviral vectors are considered a safer way to express reprogramming factors ...many passages. Ban et al developed a temperature sensitive Sendai virus which is removed by culturing cells... regulate Sendai virus transgene expression. Transient episomal delivery Non-integrating methods have...

...capacity of stem cells. Yet, very few researchers consider the sex of their cells when doing experiments—...between male and female cells. The result of not considering the sex of the stem cells was that their male... in Norway, the team realized they should also consider the sex of the stem cells. In this case, they ... many researchers I’ve talked to who’ve never considered the sex of their cells. For one thing, cells ... there are numerous methodological articles to consider—we have loaded those we are aware of in our checklist...clue or potential place to start research is by considering sex disparities in disease severity. Such disparities...checklist. A common worry for researchers wanting to consider sex is cost. Especially in animal research, they...

...Cleavage Assay Not Provided 5% Inexpensive, simple Low-throughput, low sensitivity Sanger Sequencing Provided...inappropriate for heterogenous populations, low sensitivity Targeted Amplicon Sequencing Provided 0.01%...0.01% (variable) Sequence-level data, extremely sensitive Do not sequence all DSBs, may miss unpredicted ...information from targeted amplicon sequencing is highly sensitive with detection levels as low as .01% (Hendel et... with in silico prediction to create a more comprehensive list of off-target editing events. Two of the...laboratories. Nevertheless, unbiased detection isn’t as sensitive as targeted amplicon sequencing. For example, ...throughout the genome. However, their decreased sensitivity means that the best option moving forward may...

...available with improved kinetic and sensitivity parameters. Sensitivity and Localization The calcium concentrations...are used to monitor calcium concentrations and transients in living cells and organisms. Unlike chemical...kinetics: GCaMPf (fast) GCaMPm (medium), GCaMPs (sensitive but slower). GCaMPf may be suitable for very rapid...You want to use a calcium sensor that is most sensitive to your expected range. For example, calcium in... changes with Ca while another wavelength is insensitive. Additional red sensors based on mRuby (jRCaMP1...unnecessary calcium buffering and to increase sensitivity), and is a great FRET sensor option if you have...integration within the target cells, ensuring more consistent and reproducible results in calcium assays. There...

...and potential delivery methods.   General considerations for planning your CRISPR experiment As with...experiment, there are many factors that need to be considered during the planning process. For CRISPR experiments... method: Do you need stable integration or is transient expression sufficient? Which cell types will you...are compatible with all your components. Next, consider the best form for those CRISPR components based...selection marker to generate stable cell lines. Transient or stable expression of Cas9 and/or gRNA in a ...transfer vector and used to generate AAV particles. Transient or stable expression of SaCas9 and/or gRNA. Infects...cells using microinjection or electroporation. Transient expression of CRISPR components, expression decreases...

...showed greater photostability and also matured considerably faster, with expression levels comparable to...PILATeS, Pulling-Induced Luciferase Association Tension Sensor (Zhong et al., 2024). In this new variation...and only exposed when the module unfolds under tension. When this happens, BiT and the LgBiT luciferase... Figure 5: Left: Mechanism of the PILATeS tension sensor. The LgBiT (cyan) fragment of luciferase...protein (purple) only when Titin I10 unfolds under tension. Upon binding, LgBiT and BiT Tag catalyze a luminescent...PILATeS-functionalized coverslip creates mechanical tension, and the luminescence colocalizes with the focal...lower background signal and can be used in light-sensitive systems. Most excitingly, assembly of the split...

...first-generation APEX suffered from inconsistent activity levels and limited sensitivity under the stringently low...extremely dim GFPs The availability of the three dimensional structure of GFP, its brighter mutant S65T-GFP...GFPs with diversity in fluorescence brightness, intensity, excitation and emission spectra. The lab of Dimitri... degradation. Chemical approaches may require expensive peptides or small molecules and exceed a physiologically...physiologically relevant exposure in intensity and/or duration. Both of these strategies carry the risk of...activate signaling cascades on exposure to low-intensity blue light. This approach relies on the incorporation...incorporation of light-oxygen-voltage (LOV) sensing domains from algae into chimeric Opto-RTKs. The LOV domains...

...brain. Sensitivity In ex vivo studies on mouse brain slices, nLightG and GRABNE were more sensitive than...But this first generation of tools had limited sensitivity and speed and were only available as green probes... vivo experiments. nLight indicators are more sensitive to and selective for norepinephrine and faster...that red fluorescent proteins tend to be less sensitive than green fluorescent proteins. nLightR did perform...and may soon have a red tool which matches the sensitivity of nLightG. Many thanks to Mike Waring and Tommaso...Bohacek, J., Reiner, A., … Patriarchi, T. (2023). Sensitive multicolor indicators for monitoring norepinephrine...

...antibodies when possible. If cost is prohibitive, consider an approach that prioritizes more reproducible...with representative images showing the staining intensity in the KO population (pink histogram) compared...knockout experiments can be time-consuming and expensive, it may be helpful to find an antibody that has...RNA (siRNA). Knockdown caused by siRNA is also transient, and as such, the optimal knockdown of the protein... rate. If you need a more permanent knockdown, consider using shRNA vectors instead. With both siRNA and...understand the relationship between antibody staining intensity by flow cytometry, and mRNA expression.   ... at similar levels in your cell lines, you can consider a cell treatment approach. If there is a cell ...

... This is an important downside of the technology and should always be considered when interpreting results...oligomerization of fluorescent proteins is a concern. The propensity of fluorescent proteins to oligomerize is known...brightness, photostability, maturation time, and acid sensitivity of various fluorescent proteins (Botman et al...coded by the multiple cloning site and therefore consist of random amino acid sequences or (ii) designed... an inert, unstructured peptide and therefore consisted of glycines, serines and other small, non-aliphatic...from Aequorea victoria GFP sticks out and can be considered a linker (figure 1). In fact, for several FRET...fluorescent protein fusions There are several choices to consider before generating a fusion protein, e.g. linker...

... other ligands - almost A to Z! - from ATP to urotensin. The MTRIA sensors will be useful for investigating...displays enhanced brightness, expression, and sensitivity compared with earlier SF-iGluSnFR. Image from... response kinetics and increased brightness, sensitivity, and photostability under two-photon illumination...proteins (miRFP670nano and miRFP720) and the Ca2+-sensing domain from Twitch-2B (Matlashov et al. 2022). ...plasmids here!   Torres Cabán CC, et al. Tuning the Sensitivity of Genetically Encoded Fluorescent Potassium ...LiLac has improved response size and reduced sensitivity to pH and calcium compared to previous lactate...470-495 nm) and is capable of local temperature sensing with sub-millisecond resolution and sub-degree ...

... Morgridge Institute for Research in Madison, Wisconsin. It is commonly cited that approximately one-third...modified through phosphorylation (1). However, the expansion of studies on protein phosphorylation in an array...The development of phosphoserine incorporation Extensive genomic sequencing of prokaryotic lineages revealed...specialized elongation factor that allows proper extension and translation of the pSer-incorporated polypeptide...available through Addgene, but we have worked most extensively worked with and optimized Sep-OTSλ and can recommend...the deletion of release factor 1, the protein responsible for terminating translation in response to the...between different proteins. Beyond these standard considerations, it is important to determine how much phosphorserine...

...integrating a SL-insensitive fluorophore (LSSmOrange) as an internal control and a SL-sensitive fluorophore...organoids By Ashley Waldron Organoids are three-dimensional cultures that mimic aspects of organ morphology...enzyme biosensors provide direct, specific and sensitive detection of the plant hormones strigolactones...the receptor which modulates the fluorescence intensity of the cpGFP without altering that of the internal...fluorophores, rather than on the fluorescence intensity, it provides a direct and quantitative detection...receptor-based fluorescent biosensor has a high sensitivity (50 nM-500nM range) and a high specificity for...

...temperature drops, single intensity should smoothly increase as the DNA amplicon transitions from single-stranded...detect a single copy of a transcript. But this sensitivity can sometimes cause as many issues as it solves...separate thermocycle allows you to measure the intensity of signal over an entire cycle. Since signals ...double-stranded DNA, when temperatures are high, signal intensity should be low, reflecting the presence of primarily...assumption that there is a smooth, single-step transition between ssDNA and dsDNA. This is not true for...one (Biassoni, 2014). At the bench As a highly sensitive technique, qPCR needs a steady pair of hands to...important as precision for relative quantification. A consistent 5% positive error across the plate is fine; a...

...should be consistent across all samples on the gel and should be observed as a band of consistent size and...These systems, however, require you to purchase expensive system-specific sandwiches.   Figure 2:...vendors will show that the antibody binds to transiently overexpressed protein, however, they should also...includes publications that cited the antibody, consider reviewing the materials and methods sections of...and density present in all samples. Uneven sample loading or incomplete protein transfer causes inconsistent...you require precise quantification of a protein, consider using an alternative method such as an enzyme-...

...ultimately responsible for outcomes. Be ready to support your team and take responsibility if things go...series on Management for scientists. Once you are responsible for managing others you will only be successful...you become a master at delegating tasks and responsibilities. The manager is not expected to DO all the...experience? Do you think you are giving this responsibility to someone who can do the job. It is good to...sure it goes well? As with most managerial responsibilities, good communication is at the heart of delegating...boundaries. Where are the lines of authority, responsibility and accountability? What types of issues do...

...resultant cells can either express the plasmid DNA transiently (similar to bacteria) or incorporate the genetic...ever-versatile fluorophore in a later post, so stay tuned! Transient transfection and the elusive "mammalian ORI" For... for the transfected plasmid to be expressed transiently. Since the DNA introduced in the transfection...want the plasmid depleted, but still want to use transient transfection methods. Since there are no "natural... generations. Stably transfected cells may be considered an entirely new cell line from that of the original...plasmid contains a cassette that will make cells sensitive to a toxin). In the table below we focus on positive...causes strand scissions 100-400 ug/mL *Not comprehensive.   ** In eukaryotes.   ***The concentration ...

... cells are sensitive, it might be more difficult to get them to grow out. The only downside is that it.... In theory you could use densitometric analysis to quantitate the intensity of the bands on your gel ...reagent-grade endangered Martian chicory root, but considering this is a needle in a haystack type of objective...In retrospect I used a way too high Cas9 MOI considering that A549 cells are relatively easy to transduce...stable cell pool (pool) and from individual cell expansions (monoclonal, 1-5). Wild-type A549 cells do not...followed. If you have never used this assay before, consider following their tips, including: Before digesting...

...or qPCR-based methods to greatly increase the sensitivity of detection (14-20). LDR has also been extended... do not read specific base sequences, but are sensitive to distortions in helix shape (22). Large purine... can be achieved through a combination of the intrinsic selectivity of the ligase active site and careful... that extends earlier studies through a high-sensitivity multiplexed format (21, 27). This methodology...In this method, substrate pools were prepared consisting of one target (template) strand and four upstream...Eukaryotic DNA ligases: structural and functional insights." Annu. Rev. Biochem. 77 (2008): 313-338. PubMed...JS, et al. "A high-throughput assay for the comprehensive profiling of DNA ligase fidelity." Nucleic acids...

...GCE4All: Making Genetic Code Expansion Accessible Addgene's Genetic Code Expansion Collection...protein tags. The basics These tags most often consist of an enzyme that has been mutated to block its...CP appearance at the filament tip from (a). c) Intensity traces at the tip of the filament from (a-b), ... effects on actin filament length (lower). d) Intensity and filament length traces for a different event...of organic dyes or specialized ligands is more expensive than letting the cells make a fluorescent protein...Covalent Labeling of Recombinant Protein Molecules Inside Live Cells. Science, 281(5374), 269–272. https:... W. (2011). Chemical Tags for Labeling Proteins Inside Living Cells. Accounts of Chemical Research, 44...

...turned to optogenetics. Through the use of light-sensitive protein domains, they have developed a system .... They accomplished this by inserting a photo-sensitive LOV2 domain from A. sativa into a loop region ...brain. But this process is time consuming and expensive. Thus scientists have turned to genetically encoded...electrical activity via imaging. They took the light-sensitive protein QuasAr2 and systematically mutated it....localization, has high signal:noise ratio, high sensitivity, responds quickly, is photostable, and is compatible...interaction (PPI) studies in plants that rely on transient transfection systems using Agrobacterium (such...Grefen and Blatt, 2012). The 2in1 Plasmid Toolkit consists of 4 plasmid toolkits generated with 4 different...

...recently developed a third-generation intensiometric glutamate sensing fluorescent reporter (iGluSnFR3) to...and their research.   Abhi Aggarwal: Glutamate sensing reporters Abhi Aggarwal is a postbaccalaureate ...new version of the biosensor is faster and more sensitive than its predecessors. Currently, Aggarwal, and...Research Center (DKFZ) who has developed new redox sensitive GFP2 (roGFP2)-based biosensors for live cells.... to better understand the processes that happen inside the cell,” says Pedre Pérez. Figure 2: ...changes following opioid withdrawal, providing insight into new methods for treating patients in recovery...

...within 10 nanometers of each other, or measure the tension in the helical structure of spider silk, or the...As the choice of fluorophore pairs is a key consideration for a FRET experiment, the possibilities can...Förster resonance energy transfer by monitoring sensitized emission in living plant cells." (2013). PubMed...linker sequence between the donor and acceptor is sensitive to a change in the environment which alters the...processes and signaling cascades, quantify mechanical tension (a molecular “spring”) (Meng et al., 2008), and...animals (Hirata and Kiyokawa 2016), and molecular tension microscopy (MTM), which could monitor physical ...Charlène, and Borghi, Nicolas. "FRET-based molecular tension microscopy." Methods 94 (2016): 33-42. PubMed PMID...

...for optogenetics. There are several important considerations with fabrication of the fiber optics needed...stimulation. The scatter effect is also an important consideration when preparing the implant itself, which brings...will be erratic, opsin activation/inactivation inconsistent, and behavioral results impossible to interpret...sequentially increasing fineness helps to maintain a consistent stream of light. Test your implant by connecting...least 85% of the initial laser output. The same considerations apply to the polishing and preparation of the...: closed/light receptive, open, closed/light insensitive. If you deliver a continuous stream of photons...optogenetics we’ll delve into some of the important considerations when performing optogenetics behavioral experiments...

...methodological and experimental limitations and considerations? I recently spent two months in the lab of ... at NIDA and some of the practicalities and considerations you may not find in the materials and methods...use optogenetics The most important question to consider is what exactly do you want to use optogenetics...are also available. Picking your opsin  You must consider whether you wish to stimulate or inhibit neuronal...include Channelrhodopsin2 (Chr2), a blue-light sensitive non-specific cation channel, activation of which...potential. Halorhodopsins (HR) are green/yellow-light sensitive chloride ion pumps, activation of which results...breeding, and maintenance of a mouse line is also an expensive and time-consuming endeavor. Finally, expression...

...structures, called organelles. Take a sneak peak inside a cell with the images from the Allen Cell Explorer...and diseased states can also provide interesting insights into disease mechanisms and protein function. ...complexes with. These “colocalization” studies give insights about the proximity of two proteins within the...cameras and image analysis software are highly sensitive to detecting light signals not visible to the ...human eye and are indifferent to subjective color intensity perception. Open source image analysis software... and well-executed experiments require some consideration of FP properties. It is important to look out...PMC4777285. Further Reading Microscopy U. Generfal Considerations when Using Fluorescent Proteins. Ruusuvuori...

... as one of the the skills favored by research-intensive careers. External collaborations are becoming ...important in a non-research context, especially when considering small to mid-sized companies and organizations... Addgene as a nonprofit organization is very considerate and thoughtful about its limited resources. Rather...find this to be the most challenging and time-intensive part of relationship building. However, it is ...without directly contacting them. One great way to transition from an internal to an external connection is...organization is important for success in research-intensive careers. In addition, this skill is a hallmark...Collaborate: An Imperative for Graduate Students" from Inside Higher Ed Additional Resources on the Addgene Blog...

...such as mice, which are less prolific and more expensive per individual to maintain. Also in contrast to...heart defects and cancer. These mutants are used extensively to study development and disease. Additional ...implemented in zebrafish. Developmental biology insights using lineage tracing in zebrafish To highlight...lineage tracing approaches that led to important insights in developmental biology. Microscopy-based lineage...embryo, where similar processes occur over days inside the mother, the zebrafish is an ideal system to...their long three-month generation time and labor-intensive maintenance compared to invertebrate model systems...highlight how these advantages can translate into insights in developmental biology, and advances in this...

...library/produced your lentivirus. Since viruses are sensitive to freeze/thaw cycles, we include a 1.3 mL aliquot... number of cells to transduce, you may want to consider spinfection. Spinfection is the process of transducing...transducing cells at a concentrated seeding density while centrifuging. Spinfection transduction efficiency...optimization of many variables, such as seeding density and reagent concentration.  Screening time Phew...place to ask experimental questions.  Practical Considerations for Using Pooled Lentiviral CRISPR Libraries...Libraries- A nice overview of what to consider when using pooled lentiviral CRISPR libraries. Protocols from... Waxmonsky NC, Swanson LE, Fan M. Practical Considerations for Using Pooled Lentiviral CRISPR Libraries...

...can be a time-intensive endeavor. That is why we also included an analogous set of transient constitutively-expressed...on the other hand, is a widely available and inexpensive imaging modality with good tissue penetration... be easily expressed heterologously by bacteria inside the body. So we screened gas vesicles gene clusters...bARGSer-expressing E. coli. The GV-specific ultrasound signal inside the tumors was overlaid with the anatomical B-mode...Emerald GFP. These plasmids can be used directly in transient transfection in rtTA+ cell lines (such as HEK293... using the most reliable method, which is the transient cotransfection of the mARG plamsids into HEK293T...

... the end of this post! Figure 1: Base transitions are edits between the purine bases (adenine, ...newly-deaminated inosines. Pro tip! The nucleoside inosine consists of the nucleobase hypoxanthine + a ribose sugar...transversion editor AYBEv3. This tool still retained considerable A → G editing activity at most sites, in addition... editor (gGBE) (Tong, Liu, et al., 2023). gGBE consists only of engineered MPG fused to nCas9. First, ...efficiency, purity, and indel rate of each tool vary considerably. And there’s so much more exciting protein engineering...is which. Acronym Full name Notes   Base transition Base change between purines (A ↔ G) or between... glycosylase to a CBE; the first type of base transition editor invented.   Abasic site A DNA site with...

...” in quotations here because I would not have considered myself neutral in any of their disagreements....Skills series, this post is not meant to be a comprehensive guide to Conflict Resolution. There are countless..... Ownership: Misunderstandings about who is responsible for what tasks or projects is a common source...customer support element amongst their other responsibilities. Knowing which aspects of customer support... require a manager to explicitly assign the responsibility in question to a specific person or team. ... more of a winner and loser–one party will be considered in the right, and one in the wrong. If you arbitrate...also aggravate long-term personal conflicts.  Transitioning a conflict into a collaboration may not be the...

...primary antibodies is a time consuming and expensive process. Consider the vast number of possible primary antibodies...we’ll go through some of the most important considerations to make when planning and performing an IF ...indirect immunofluorescence procedure with key considerations about each step. Image created with BioRender.com...’ll start there. Some of the most important considerations are to make sure your secondary 1) will recognize...remember that antibodies recognize very specific 3-dimensional epitopes. Different sample processing steps can...protocols available for each antibody that you consider in order to determine how reliable the antibody...

... imaging NIR FPs, consider using light sources that produce enough light intensities in NIR. Usually, ...supplied (Baloban et al., 2017).  It is important to consider effective brightness of NIR FPs in cells, in addition...Shemetov et al., 2021). In addition to FRET, intensiometric calcium indicators NIR-GECOs were developed...dDetermined as effective NIR fluorescence in transiently transfected live HeLa cells with no supply of...Designing brighter near-infrared fluorescent proteins: insights from structural and biochemical studies. Chem ... (2015). Piatkevich, K.D. et al. A robotic multidimensional directed evolution approach applied to fluorescent...

..., for some applications, integration might be considered a double-edged sword with unintended consequences...AKA transduction in the viral vector world). Once inside the host cell, the viral payload is integrated ...lentiviruses lack a functioning integrase, the enzyme responsible for inserting viral DNA into the host genome....genomes are transcriptionally active, leading to transient expression of gene products (Wu et al., 2004)....insertional mutagenesis (Howe et al., 2008). 2. Transient expression of transgenes/shRNAs If you want your...expressed (Fu et al., 2013). Using IDLVs ensures transient expression without the risks associated with prolonged...of clinical or basic research applications. Considerations for IDLV use If you’re reading this and thinking...

...results of cloning or knockout experiments to forensics and clinical diagnostics. One example of southern... a sample. Whereas Southern blotting is labor intensive and requires a large amount of high-quality DNA...lacking the 3’ hydroxyl group required for the extension of a DNA strand (8). Adding a small amount of ...of DNA strands of all possible lengths because extension is terminated upon the incorporation of a ddNTP...speed ultimately leading to what are generally considered first generation DNA sequencing machines (9).... drive for faster, higher throughput and less expensive sequencing fuels a sequencing revolution that ...1195397. 2. Chang, Judy C., and Yuet Wai Kan. "A sensitive new prenatal test for sickle-cell anemia." New...

...include a Packaged on Request option. Through this expansion, scientists will be able to request an AAV composed... vector prep.    Excited? So are we! This expansion will give researchers increased flexibility when...overview in this blog post.     When will the expansion happen?  You will be able to request Packaged ... Plasmids available for Packaged on Request consideration will have a banner on their material page. Interested... in-stock preps. If a request has technical considerations that may affect expected titers, a customer...resources.  We hope you’re as excited about this expansion as we are! Happy packaging! ...