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...Cas protein variants may also be useful for targeting ncRNAs. For example, the Cas12a variant can cleave...RNAs in Plants with the CRISPR-Cas Technology is a Challenge yet Worth Accepting. Front Plant Sci. 2015...transcribed but do not encode proteins. They play important roles in many cellular and regulatory processes...into the break sites. Knock-in experiments are important for visualizing ncRNA localization and function...complex and not yet fully understood, so it is important to carefully select the target locations of knock-in... ncRNAs.  CRISPRi and CRISPRa For scientists who want to modulate ncRNA expression without permanently...genomic elements (Hazan and Bester 2021). Other Cas variants Another option is CRISPR/Cas13, which targets ...

...COVID-19 research in other labs. Spike protein, variants, and pseudotyping The SARS-CoV-2 virus depends...cellular receptor. To more easily study the spike variants, scientists have developed ways to pseudotype ...part of a study that examined the repertoire of antibodies from 19 COVID-19 convalescent individuals (Tong... has had many opportunities to evolve. Several variants are now circulating and plasmids containing these...pseudovirus and those encoding different spike protein variants that can be incorporated into pseudoviruses. The...at neutralizing the different SARS-CoV-2 spike variants. You can learn more about their research in this... COVID-19, aside from the widely used qPCR and antigen tests.  Labs including Jennifer Doudna’s lab and...

...nucleoporin. Lu et al. Plant J. 2010 Yuhai Cui Red mScarlet Split-wrmScarlet variant of mScarlet Split-wrmScarlet...Complementation (BiFC). It can provide a qualitative or quantitative readout of the interaction in vivo or in vitro...includes I152L in N-terminal fragment An enhanced recombinant amino-terminal acetylation system and novel in...aa 155), includes I152L in N-terminal fragment Constant rate of p53 tetramerization in response to DNA...–10) and sfCherry2(11), also photo-activatable variant PAsfCherry2(1–10) Improved split fluorescent proteins...presence of heme oxygenase for chromophore formation (abundant in eukaryotes) A Near-Infrared BiFC Reporter for...presence of heme oxygenase for chromophore formation (abundant in eukaryotes) Bright monomeric near-infrared ...

...portable quantification of diagnostic results.  Our approach now detects samples at clinically relevant concentrations...in a clinically relevant setting. Molecular diagnostics for the Zika virus predominantly look at viral ..., and North America. Diagnostics will play an important role in helping to monitor and slow this spread... synthetic gene networks for the detection of antibiotic resistance and strain-specific diagnostics for...develop our paper-based platform into clinically relevant diagnostics for the Zika virus was daunting. It...American-lineages). Strain discrimination is especially important in the current outbreak because of the neurological...Pardee and Alexander Green! Keith Pardee is an Assistant professor in the Leslie Dan Faculty of Pharmacy...

...applications. As individual libraries can vary significantly, it’s important that we have an amplification protocol...and the desired coverage for the library (How important is it that every single plasmid in your library...protocol. Created with BioRender.com.   The most important factor to consider is your desired library coverage... plasmids in your library. As an example, if you want 100x coverage of your library, and your library ...500,000 colonies.  Pro tip! Since you probably do not want to individually count 500,000 colonies, you’ll likely... is less than that of electroporation, but an advantage is that chemical transformations are generally...overtake larger plasmids, if the library varies significantly in plasmid size). On plates, each transformed...

...glass cloning rings to isolate colonies of antibiotic-resistant cells; during the time required to grease... video below) is simple: After obtaining antibiotic-resistant colonies, use a Sharpie to circle 12-48 ... do not normally use antibiotics. Lastly, and perhaps obviously, it is important to have a functional ... inside of the wrist or the cheek. (Parents of infants will have no problem with this!) Make sure your...levels of expression of our gene of interest. Advantages of the agarose cloning method The agarose technique...cells did not seem to form discrete colonies but constantly migrated away from each other. In our experience...morphology or other physiological properties. An advantage of this technique is the potentially large number...

...he didn’t want to spend hours reviewing papers to piece together the best protocol;  he wanted to get going...how to prioritize but I  just added the “most important” tasks my schedule. This was bad for 2 reasons...to focus on what you should be doing : writing a grant, thinking about the next project, reaching out to...expectations, answer any questions, and (most importantly) decide on a timeline. My experience is that ...your team’s success depend on it.  Equally as important is to be able to actively listen - this can be...working with X?”  is going to get you what you really want to know. Remember that one on one discussion can...counter-productive. I always assume that reports want to succeed at work and providing feedback is needed...

... the participants had certainly realized the importance of seeking out mentoring as an important aspect...Kind of Mentor Do I Want To Be?” In this excellent essay Acclimatrix, an assistant professor at a research...current mentors/advisors like Yoda? If so, you might want to find additional sources of wisdom. While somewhat...thoughtful treatise on the kind of advisor/mentor she wants to be for her new lab. If only every science trainee...dedicated person guiding the start of their career. “I want to be a PI that holds my students accountable for...being coddling, to listen without being taken advantage of, and to respect my students as developing colleagues... If you have a question about mentoring that you want us to cover, let us know.   ...

...skills. Why are transferable skills so important? It is important to identify which transferable skills...means you’re guaranteed to get the job, however, many times the hiring team will also want to examine not...of demanding courses, a capstone paper I really wanted to hit out of the park, part time work, bills, ...through school, jobs, and volunteer work, and I guarantee you have developed them through similar experiences...potential employers, collaborators, or co-workers want to know that you are a well-rounded individual with...skills, before offering someone their dream job; They want to know if the candidate has what it takes to thrive...ask about your creativity, your potential manager wants to hear a specific time that you thought outside...

...probably also very interested in having high-quality antibodies and reagents that you know work…preferably before...not alone – we’re all looking for high-quality antibodies and reagents that work for our experiments!) ...critical for the performance of a reagent, e.g., antigen retrieval conditions, dye inactivation details,... the Human Biomolecular Atlas Program (HuBMAP) antibody metadata standards. And the dataset is rich...tissue preservation, target tissue, detergent, antigen retrieval conditions, and more. Users can also ...seeking to create an iterative process that is constantly becoming better, with more data, more discussion... a great place for anyone who has used IBEX and wants to preserve and share their validation work. Everyone...

... in a creative problem-solving process?  You may want to start by carving out time to tackle it. It’s ...problem. In a study outlined in Originals by Adam Grant, a creativity expert at Stanford conducted an experiment... ideas heard.  So how can we assure that all participants feel comfortable sharing their ideas? And how...encouraging dissent, the next tip may be particularly relevant to you. Elect a devil’s advocate. This is the ... with the group (because they will fight more adamantly for their solution), selecting a devil’s advocate...implemented, impacted the entire company. It is important to note that the person who submitted the idea... well received, understand that people can be resistant to change. Ask your peers or your superiors to...

...might want to optimize codons for expression in a particular organism. Why is codon usage important? Translation...Ultimately, it is important to take a close look at your experiment and gene that you may want to codon optimize...what does this mean for a molecular biologist who wants to express genes from one organism in another? Let...polypeptide chain. One part of the tRNA contains the anticodon,  a three nucleotide sequence complementary to... binds the amino acid that corresponds to the anticodon sequence. That means there are 61 possible tRNAs...tools to help with these decisions.  So when do you want to codon optimize your gene of interest? Codon optimization...not naturally express that gene. The protein you want to express may contain codons that are rarely used...

...position do you want your gene to be in relation to the rest of the genes? And do you want it to be in the...This post was written by Alfonso Timoneda, with significant contributions from Robert Hurt and Mohamed Soufi... perfect Our first tip, and arguably the most important, is to take some time to sit down, plan your cloning...a clear vision of the final vector structure you want to achieve before starting your experiments. Don...sequence, a terminator, or other plasmid part? Do you want to fuse your protein to any tags or fluorescent ...MoClo kit through their online catalog pages. We guarantee this will save you a lot of headaches down the... start your cloning, it is advisable to remove unwanted type IIS restriction sites within your sequence...

...popular job site webpages. Importantly, find a good mentor that can share relevant experience and resources...what comes next. To that end, one of the most important things that you can do now is diversify. Read ... position! Conferences One of the single most important things you can do to plan for your career is to... Speaking of jobs, it seems like an obviously important experience, but far too many students and young...stand out with key words and by putting your most relevant experience at the top. You may also consider visiting...opening, you can send your contact an email with the relevant info and they just might personally hand your ...can search for jobs, post your CV, and, most importantly, manage your professional network. This is probably...

...growth medium. Induction timing is important, as you typically want to make sure your cells have first...coli strains are not usually well suited for recombinant protein expression. Many challenges can arise...coli. We will review the potential pitfalls of recombinant protein expression and some of the most popular...or are enzymatically active. For this reason, recombinant proteins are typically expressed in E. coli engineered..., which reduces proteolysis of the expressed recombinant proteins. lon protease: Strains where this is...pLys strain. Your strain may require additional antibiotic selection or a special growth media, or if your...your plasmid is low-copy, consider reducing the antibiotic concentration. Growth Tempurature: Analyze your...

...employment, so it’s important to make sure a masters helps you get a job you want. But how do you know...I didn’t plan to get a master’s degree. I wanted to be a scientist and you need a PhD to be a scientist...prepared me to be exactly the type of scientist I wanted to be. But prior to graduate school, I wasn’t sure.... Don’t discount job postings looking for PhD applicants since many will also accept applications from...your work at conferences, help with manuscript and grant preparation, and lead your own research project.... lend itself more readily to a switch. 4. Do you want more autonomy?  Before my master’s, I worked in ... over. Everyday was the same and I was bored. I wanted more variety and more power to influence the direction...

...IgG) thus targeting MNase to antibody bound proteins (Figure 1). Importantly, since Protein A has weaker... culture, because it is easy to harvest large quantities of cells. However, when working on specific tissues...Washington. CUT&RUN doesn’t require fixation and significantly decreases background noise, reducing the read...dissections. With CUT&RUN, we have had success with significantly fewer wings, such that enough tissue for an ...beads. Nuclei are then incubated with a primary antibody against the protein of interest. The Protein A...is helpful to incubate with a rabbit secondary antibody when using a non-rabbit primary, prior to the ...improvements to the initial CUT&RUN protocol, the most significant being the usage of a strong detergent to permeabilize...

...its advantages, most scientists still encounter several struggles with these techniques: unwanted mutations...chemically competent E. coli and transformants selected with the appropriate antibiotic. Although the precise mechanistic... isolation, insertion and amplification of a recombinant DNA without sequence alteration. Molecular cloning...overhangs, insert and vector purification, and, most importantly, excessive time requirements. FastCloning is ...ends. See an overview of the PCR process and the resultant products of the reaction in the figure from Chaokun... technique there is a chance of introducing an unwanted mutation. Tips for high efficiency cloning Now...Annabel HA Parret, and Anastassis Perrakis. "Recombinant cloning strategies for protein expression." Current...

...grateful to the grant officers at Fast Grants. We received a miraculous grant from Fast Grants, which was ... plasmids and to receive important coronavirus-related deposits. Our valiant lab and shipping staff agreed...collaborations as well as provides visibility for grant-driven research being done in all labs sharing materials...materials via Addgene. Perhaps if each of many grantors supporting research could provide Addgene with... for supporting Addgene by providing a generous grant. This support will enable us to continue all operations...

...produce recombinant proteins at high levels. In a case where clinical applications of recombinant proteins...most commonly used line expresses SV40 large T antigen and is thus dubbed HEK-293T. Given that the modern-day...HEKs. These systems built around HEKs offer an advantage to use the cell line and we will highlight some...production because they express the SV40 large T-antigen. This allows them to replicate viral plasmids with...viral particles are then filtered to remove any unwanted material (HEK cells, etc.) and are ready to use...also be adapted for serum-free growth, which significantly reduces the cost of production. Research applications...gene expression, growth rates, etc.). Thus, it’s important to maintain short culture periods (less than 20...

...said. “We know CREB is really important, but where is it really important and when? That is not known.”...injected, and they don’t always end up where you want them to go. Griesbeck is motivated by a particular... adds that this color change has a particular advantage for observing events taking place inside cells...from muscle cells. They identified a toadfish TnC variant which had a high-affinity for calcium binding and...Methods, the FRET changes of these Twitch sensor variants were optimized by testing various linker configurations...large-scale functional screen in bacteria. Sensor variants were then refined by a secondary screen in rat...“trick” to the development of additional color variants. This resulted in the construction of redCALWY...

...Addgene’s Science Video Production Specialist, I’m constantly thinking about ways to improve our productions...going to be out of the office for longer than anticipated, we had to think of something else.   ...talking to their webcams.  Build: I sent the participants interview questions they could answer, and I...directing. Everyone’s schedules were busy, so it was important for me to let people record at their leisure. ...for our other videos. For starters, I hadn’t anticipated nearly every webcam recording only 1280x720 resolution... were still many restrictions in place, most importantly, masks.  Besides the muffled voices beneath masks...with them. I knew from my previous cycles that I wanted to be able to talk to people and direct them. For...

... were corrupted by unwanted indels on the same allele, probably due to concomitant NHEJ repair. At the... cells, HDR is relatively rare and DSBs are predominantly repaired by NHEJ. While other groups have focused... locus, over 90% of the HDR edited alleles had unwanted insertions/deletions, therefore making those alleles...is accurately edited, the other likely contains unwanted indels. Following this observation, we tested ...introduction of an intended mutation while taking advantage of the improved efficiency provided by blocking...outcomes and in many cases still didn’t get what we wanted. The Indels were gone from most HDR reads that ...to direct the zygosity of the cells. This was important for us because the Alzheimer mutations we study...

...contributed by guest blogger Joachim Goedart, an assistant professor at the Section of Molecular Cytology...fluorescent protein (GFP) from Aequorea victoria and its variants are genetically encoded fluorescent probes. One...Organized Smooth Endoplasmic Reticulum (OSER) assay (Costantini et al., 2012) measures homodimerization of FPs...the appearance of OSER whorls. This assay is quantitative and is currently the standard for assessing ...2016; Lambert, 2019). These characteristics are important, but have less priority than stickiness. In my...lead to over-expression artifacts. This is an important downside of the technology and should always be...linker length, the specific fluorescent protein variant, and where to add the fluorescent tag. These choices...

...format meant that the posters could be viewed publicly and stored in one place if the participants used ...involvement and how much time you want to spend with the conference. “If you want to have the same experience...first sent a pre-workshop questionnaire to the participants to learn about what they were looking to learn... content. In the middle of the workshop, the participants took part in a protocol drawing exercise. The...workshop also used a shared note-taking doc where participants could share their notes together. Figure...session you’re presenting is eye contact. “It’s important to keep your video on and look into the camera...there are many creative ways to adapt and take advantage of the new conference format. Additional resources...

.... Sacks, Musicophilia includes case studies of relevant neurological atypicalities, including a patient...and fascinating, all without compromising on the relevant science."     - Randall Carter, Contracts Administrator...Microbes are everywhere and this book emphasizes the important roles microbes play in our ecosystem, examining...relationships between microbes and insects, mammals, and plants, among others. I particularly enjoyed the emphasis...series is the first 3 books of a larger epic of fantasy novels. Set in a world where 1000 years ago the... Norrell by Susanna Clarke "A quasi-historical/fantasy fiction. Two dueling magicians in the 1800s in ...launching this one. On a purely selfish level, I wanted to be challenged to read some books outside of ...

...the controversial medical issues. Participants gave significantly more favourable ratings to the abstracts... none of the participants had preconceived opinions about “adeolitis.” The participants in my study were...biochemistry, microbiology, epidemiology, and bioanthropology. Studying human health science made me happy...approached me for advice on her baby’s health but adamantly refused to get them vaccinated. A classmate told...told me that they never finish a course of antibiotics, choosing instead to save a few days’ worth of ... affirm or challenge their beliefs. I asked participants to read short abstracts comparing the mortality...texts were otherwise identical. Likewise, when participants read an abstract for a study with findings that...

... your least abundant proteins, with the blue and infrared channels used for more abundant proteins or ...compatible with your microscope optics, you will want to compare the excitation and emission spectra of... microscope. Ideally, you would like to have substantial overlap between the excitation and emission filters...lecture at iBiology. Commonly used filter sets & relevant fluorescent proteins Commonly used filter sets... are mTagBFP2, EGFP or one of the improved GFP variants, mRuby2 or TagRFP-T, and an infrared fluorescent...In mammalian cells, one of the improved folding variants of EGFP like mEmerald or Clover is probably best...measurements. Here, EGFP outperforms the improved folding variants, presumably due to the lower growth temperature...

...available. Table 1 compares some general advantages and disadvantages of each approach. Table 1: Properties...nighttime displays, these insects have contributed an important tool to the scientific community: luciferases,...levels and improved signal sensitivity. Another advantage: luciferase reporters aren’t susceptible to photobleaching...Japanese firefly (Luciola cruciata), and more. These variant proteins offer different kinetics, substrate requirements...luminescent substrate. This approach preserves the advantages of luminescent assays (low background, no photobleaching... that produce different colors or have other advantageous properties. This approach can improve live-cell...measurements of luminescence from live cells. Luciferase variants with incompatible substrates can also allow multiplexing...

...macromolecule density is equal to the buoyant force of the solution. Contaminants of lower densities are captured...Iodixanol Gradient Ultracentrifugation First, you’ll want to identify the reader’s goal. Why are they reading...What is the goal, who is my audience, and what do I want to be different for them when I’m done? What are...wait!  Done? Excellent! We asked our workshop participants to do the same thing, and even though only about...between layers are muddled due to the passage of supernatant through each layer. The right panel illustrates...before and after ultracentrifugation. The viral supernatant migrates through increasingly dense layers until..., and we’d love to hear yours as well.  More importantly, you can use these four steps for any type of...