...structures or other local genomic elements (Hazan and Bester 2021). Other Cas variants Another option is CRISPR...ncRNA (Jiang et al. 2014).  Simple knock-out experiments are trickier when it comes to long non-coding... et al. 2018). However, removing large lncRNA fragments with multiple gRNAs poses the risk of disrupting...can be minimized by disrupting lncRNA regulatory elements that are crucial for transcription and downstream...or introduced, into the break sites. Knock-in experiments are important for visualizing ncRNA localization...long ncRNAs is impractical. Instead, tags or stop elements can be inserted to disrupt the transcription of...CRISPR/Cas9 system is not effective for your experiments, there are plenty of other CRISPR-based options...

...only will these mStayGold variants be valuable tools for your next experiments, they will also likely inspire...genome editing for primary cells Monomeric StayGold variants AAV.CAP-Mac for systemic gene transfer in primate... Breaking up is hard to do: Monomeric StayGold variants By Mike Lacy In 2022, Atsushi Miyawaki and colleagues...stability. Here’s a quick overview of these new variants:   Figure 2: Inner mitochondrial membranes...The Miyawaki lab generated a series of StayGold mutants and identified a triple mutant, Y187F, R144I and...Addgene here!   Ando, R., et al. (2023). StayGold variants for molecular fusion and membrane-targeting applications...gene delivery capsids previously identified in rodents have not been widely successful in primates. The...

...may undergo genomic rearrangements during replication. To avoid genomic rearrangements, try amplifying the...contact A variety of reagents can increase transduction efficiency. These reagents typically enhance the...working with virus: 1. Viral vectors undergo DNA rearrangements. Depending on the composition of the viral...viral vector with bacteria designed to minimize rearrangements such as NEB Stable or use a standard strain...ensuring viral production is to perform PCR against components of the viral vector using the harvested viral... you time, and allow you to standardize your experiments later. Stay tuned to the Addgene blog and be ... pages to get up-to-date tips for your virus experiments. References 1. Anthony, Simon J., et al. "A...

...to extract without harsh reagents and patience. Plant expression systems Plants provide a cheap and low-tech...through the use of protein tags and separated from contaminants using a variety of purification methods. Deciding...systems can be scaled up for production of large amounts of protein. Some drawbacks of insect cell expression... systems. Bacterial expression systems When one wants to produce vast quantities of protein rapidly and... DNA vectors that can be used to produce large amounts of protein in bacterial cells: the pET, pRSET, ...recombinant proteins. Many cells from various types of plants such as maize, tobacco, rice, sugarcane, and even...share many of the same features and processing requirements as mammalian cell expression systems, including...

...repetitive genetic elements that are found in both prokaryotic and eukaryotic genomes.  Components of a transposon...around in the lab.  Let’s take a look at the key components of transposons, how they are classified, and ...target sites. Integration of most transposable elements (TEs) results in the duplication of this target...insertion.  Types of transposons Transposable elements are split into two classes based on their mechanism...the rest of this article. Class I Transposable elements (TEs): Retrotransposons  Class I TEs are also ...screens Transposons, by their nature, are mutagenic elements, which makes them a great tool for mutagenesis...developed the INTEGRATE (Insertion of Transposable Elements by Guide RNA-Assisted Targeting) system. INTEGRATE...

...Contamination by humans accounts for the largest source among those mentioned above. The contaminants can spread through...antibiotics and are broad-spectrum antibiotic agents. These agents can be used to wash the cell cultures. The...to the liquid or semi-solid medium (containing nutrients essential for mycoplasma growth). Infected cell...synthesized by eukaryotic cells. First, the kit components rupture the mycoplasma and then specific enzymes...for the lab to avoid bringing in environmental contaminants.  Figure 2: An Addgenie works in the...Photo by Kate Harten DeMaio. Cell lines and reagents Examine your cell line stock for mycoplasma contamination... subculturing. Avoid using expired products or reagents that have been stored for a longer time. Use fresh...

...DNA you’re purifying (plasmid, genomic, or DNA fragments from agarose).    In this article, we will go ...or agarose gel slices Product: plasmids or DNA fragments The plasmid purification and DNA gel extraction...Starting material: agarose gel slices Product: DNA fragments You might be wondering, “How is milk going to ...or even paper towels to purify DNA and RNA from plants, microbes, blood or mammalian cells in 30 seconds...into this mixture to absorb the nucleic acids. Containinants bind cellulose less tightly than nucleic acids...placing the dipstick into a wash solution removes contaminants while nucleic acids stick around. Finally, purified...gel slices Product: purified PCR product or DNA fragments Maybe you’re not ready to give up DNA purification...

..., case studies, and experiments for labs, in addition to testing those experiments. “We also do videos... with students and faculty, Caitlin also collaborates with the sales and operations departments at eScience... on her job as an SME: “I write curriculum for students taking science courses online, which is a blast...then condensing that and making it accessible to students.” Making science approachable is often the most...in isolation. “A lot of what we do is work with students who don’t have a science background. [We ask them... demonstrating an experiment or a technique to students, like how to pour a gel [for] electrophoresis ...team to meet faculty and introducing the kits to clients who might initially be doubtful of online science...

...analyzing thousands of edits performed over multiple experiments and comparing the robustness, accuracy, and speed...also provides a Knockout score (KO-score) which represents the proportion of cells that have either a frameshift... How ICE works   Following delivery of CRISPR components into target cells, genomic DNA from both edited...analysis, which can compare up to five editing experiments at a time, and batch analysis, which compares...confident you can be in the ICE score. KO Score - Represents the proportion of cells that have either a frameshift...the edited pool. The black vertical dotted line represents the cut site, and yellow + symbol on the far ...trace. The horizontal black underlined region represents the guide sequence, along with the red underlined...

...permeabilization with detergents such as triton-x, saponin, or tween. Take care when using strong detergents like triton-x... and electronics systems, FACS instruments have specialized components that can divert a subset of cells...overcome these challenges and make these types of experiments easier and faster to perform. Introduction to...channel number based on intensity. Higher intensity events are assigned to higher channels. In a typical assay...assay a user will compare the shifting intensity of events. Since intensity positively correlates with expression... flow cytometry particularly well to complex experiments looking at a variety of different targets. If...for setting up antibody based flow cytometry experiments.       References and resources References:...

...immunity in diverse tissue environments. These studies provide quantitative measurements of cellular motility... tissues structures are more stable and local movements predominate (e.g. a basal progenitor cell migrating...proteins can and have been used to monitor cellular movements in the immune system. The techniques used here... Studying cell mobility within these complex environments has never been easy and, for many years, researchers...to count cells and statistically analyze cell movements. By using cells labelled by fluorescent chemicals...giving researchers the ability to monitor cellular movements in real time. This technique was first Introduced...use of fluorescent proteins to track cellular movements and interactions. Even if virtually all fluorescent...

... are key elements of an AAV plasmid. Parts shown in grey are optional cis-regulatory elements.   Key...sequences. Other cis-regulatory elements Next let’s talk about some of the elements that aren’t required parts... common AAV plasmid elements! And while there are many other AAV plasmid elements and combinations out... Where do you start? Turns out like all good experiments, making AAV starts with some plasmids. You just...Key elements of an AAV plasmid First let’s talk about the key parts of an AAV plasmid. Inverted terminal...Addgene blog Learn more about basic viral vector elements Read about four considerations for the perfect...

...OCRL1 gene that leads to the early death of young patients as their kidneys fail. The condition was first...piqued their interest. They obtained cells from patients with the condition to reveal some “outstanding...problem. “We’ve had the opportunity to meet a few patients that suffered this disease,” Aguilar said. “Once... (XP). This recessive genetic condition leaves patients incredibly sensitive to sunlight and prone to ...interested in XP out of a desire to understand the early events in the transformation of healthy cells into melanoma... with CRISPR-Cas9. There are so many different variants and strategies out there. This will help us acquire... as Alternating Hemiplegia of Childhood (AHC). Patients affected by AHC suffer from recurrent attacks ...

...cell lines. As I started planning out my own experiments in graduate school, I had to divide my time between...the long and highly useful tasks. The quick accomplishments will likely energize you. For example, let'...experiment fails, you’ll still have made small accomplishments each day that should help keep you content ...section below, it’s important to dedicate specific amounts of time to specific tasks beforehand and assess...my undergrad days, I had general goals for my experiments, but didn’t spend enough time thinking about ...quite difficult. In my experience, planning has 3 components: A mechanism for blocking off time for different...make sure I have explicit goals in mind for the endpoints of a task or project (with metrics if possible...

...homologous DNA fragments in a single-tube reaction. For mutational scanning experiments, libraries of ...of a plasmid library that expresses all desired variants of a protein. Applying a selective pressure winnows...winnows the pool down to plasmids expressing variants with optimal function. High-throughput DNA sequencing...mutational scanning Hundreds of thousands of protein variants can be tested in one experiment. There are many...of DNA fragments that contain variable bases (NNN) at any given codon site of interest can be cloned into...deficient in dUTPase and uracil deglycosidase. dUTPase prevents the buildup of dUTP and uracil deglycosidase removes...across a region of interest, generating multiple mutants in one PCR reaction. Below is a step-by-step overview...

...all the variant PUF segments required to bind any 8 base RNA sequence. The segments are provided in Gateway... have any questions, please write to us in the comments section.  FX cloning kit This kit is designed...Translation of the standardized leader peptide prevents secondary structures in the mRNA encoding your...expression systems; the Cell-free expression test kit represents a set of 88 clones in a pCellFree vector that...quantitatively determine allele frequencies across timepoints or populations. This kit consists of a 48 plasmid...ordered progression to resistance." Antimicrobial agents and chemotherapy 57.11 (2013): 5373-5383. Walkiewicz...Assembly Kit PUF proteins are composed of repeat segments that can be engineered to bind specific RNA bases...

...condition 1” points (POI and loading control) are smaller than the analogous green “condition 2” points by the...blot.   Total protein measurements as loading controls Total protein measurements are an accurate alternative... example gel (left) with bands corresponding to points on the plot of protein abundance versus band intensity...the exact same value. For the two solid POI points, this difference is reflected on the band intensity...intensity axis. But the loading control points are outside of the linear detection range. As a result, they have...Outside the linear detection range, band intensity measurements become extremely susceptible to error, since...for your POI. Figure 6: Total protein measurements are taken on the same gel or membrane (depending...

...fluorescent proteins, with recombination events specified by three loxP variants, but the principles remain the ...recombination to generate additional recombination events, and the synthesis of alternative loxP sites to...expression; these deletions are defined using 2 loxP variants (only identical loxP sites can mediate recombination...either deletion removes one of the other loxP site variants, preventing further recombination. Brainbow-1.1...) The construct contains two tandem invertible segments, each with two fluorescent protein coding sequences...combinatorial effect of multiple, independent recombination events leads to a rainbow of colors. With three copies...Variations Between Brainbow Systems   Brainbow variants and applications beyond the mouse brain In addition...

...same output: a spreadsheet of read counts. You can use these counts to confirm the representation of your...when you need to amplify a library for your own experiments. Library amplification 101 Library amplification... lots of library DNA is needed for screening experiments, so the scale of the DNA prep is larger. And ...is similar for most libraries. Here are the key points you’ll need to remember when undertaking library...enough DNA and competent bacteria to ensure all components of the library are represented. Typically, Addgene...need to count how many times each of the library components is found in the read list. If you also sequenced... your library. When plotted as a histogram, the counts will give you a bell curve of the abundance of ...

...undervalued.” Connie Cepko points out the limited choices in hiring graduate students and postdocs when starting...be half of my day sometimes.” He points out that the copious amounts of email are probably because a PI... than just experiments and grant writing. You have to enjoy the mentoring role.” She points out that new... having projects ambitious enough that everyone wants to recruit rather than repel and hoard.” If you'...it is once it gets fully up to speed.” He also comments that it is also surprising “ how easily people...last post in this series “Fun in the lab!”   Acknowledgments Thank you to Dr. Tom Ellis (Imperial College...