...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...

... 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...

...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...

...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...

... 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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

...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...

.... 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 ...