...vector remains episomal, meaning it stays separate from the genome. One option for episomal vectors is to...prokaryotic ribosome, while G418 (geneticin) is effective at inhibiting the eukaryotic ribosome. One resistance...will be lost from the population. A bacterial chromosome has a single origin of replication (ori), where...lines that express viral machinery. Alternatively, some slow-growing cells, particularly mammalian cells...but it won’t allow you to select for your vector. Some antibiotics block important cellular pathways shared...across kingdoms by targeting a shared aspect of ribosomal function. A puromycin resistance gene on your ...vector to survive while the auxotrophic cells die. Some auxotrophic selection markers can be used across...

...best for you. Some of these important questions will be easy to ask the lab head, and some are better asked... the right lab such a big deal? It’s actually something you CAN choose and it will make a huge difference... level of attention and interaction (and I did). Some of the information you will need is about you. It...before making this important decision. I’ll highlight some of these questions in the sections below. Mentoring... help identify your priorities and needs, but sometimes it just takes introspective thought on what’s ...responsibility to train you seriously. Why work for someone who you don’t look up to and who doesn’t care about...performance and learning? Everyone’s different so give some thought to what atmosphere makes you successful....

...alleles. Some alleles, such as Cre, function when hemizygous, meaning that only one chromosome needs to...randomly into their genome, some transgenic strains are difficult to breed. Some problems with transgenic... if the mutations are on the same chromosome. Homologous chromosomes recombine during meiosis, creating...homologous chromosomes in the parental generation, they are likely to stay on separate chromosomes in future...distance between the two loci of interest on the chromosome, the less likely crossing over is to occur between... which would get both mutations onto the same chromosome. Until crossing over occurs between the two loci...

...integrity. (Not the case, as I’ve discovered.) As someone who has now seen both sides of the postdoc experience...decision a little easier if an industry postdoc is something that you’re considering. Like anything, there ...can’t afford to spend too much time working on something extremely risky or exploratory. Getting a job ...experience” that I’ve seen listed as a requirement on some industry job postings. Also, if you work in industry...their pipeline projects. In this scenario, finding someone with whom you can have an in-depth discussion about...Genentech, among others, and the list is growing. Some post positions as they come up, whereas others post...companies. Like most jobs, it does help if you know someone who’s already in industry to land you that position...

...’s no genomic integration Replicating EBNA1 episome Non-integrating Medium Medium Generates transgene-free...integration Inefficient Need to verify loss of episome Minicircle Non-integrating Medium Medium Generates...-deletable or Tet-inducible lentiviruses address some of these concerns, but overall integrating viral...for translational use. Viral Vectors Retroviral: Some of the first iPSCs were generated with Moloney murine...cut and paste” mechanism between a vector and chromosomal DNA. PB has a cloning capacity of ~9-14 kb. This... Sendai virus transgene expression. Transient episomal delivery Non-integrating methods have a smaller...oriP/Epstein-Barr nuclear antigen-1 (EBNA1) based episomes: These plasmids carry the origin of replication...

... come across a couple other terms that can cause some confusion. Paraformaldehyde: Many IF protocols ...your sample, cross-linking can block the epitope of some targets, preventing your primary antibody from binding...antibody binding by tweaking those parameters, but in some cases your antibody may just not be suitable for...organic solvents may improve antibody binding for some targets, they can destroy it for others. That said...tough-to-permeabilize membranes and these detergents avoid some of the negative effects caused by organic solvents...BioRender.   How do I choose?! Ok, so now you know some of the possible types of fixatives or permeabilizers...but how do you decide which ones to use? Here are some tips for making those decisions: Always always ...

... similar protocols, and look - they’re seeking someone who has, to a t, all the research skills under ... but also transferable skills, before offering someone their dream job; They want to know if the candidate...skills.” It is nice when other people point out how awesome we are, but there is also value in knowing and ...prepared for new and exciting opportunities. Here are some strategies for identifying your transferable skills... and using your charisma to give people hope for some shiny vision on the horizon. You’re about to keep...happens at your organization, when your boss needs someone to step up, when a new venture presents itself ...feedback, bask in the glow of a job well done, take some time to think, and write your thoughts down! What...

...discussed in Chapter 11)? Even if some of the problems are addressed in a somewhat cursory manner, their inclusion...Zimmer to introduce any one of these topics without some editorializing, and he wisely makes no such attempt... is, presumably, why they ended up in this book. Some of these issues are not particularly new. Chapter...science would be complete without the inclusion of some interesting case studies, and Zimmer provides several...sections on optogenetics and CRISPR of course address some of the ethical implications of using these technologies...changing nature. Full disclosure: Zimmer also spends some time on Addgene’s role in the rapid dissemination...suggestions are not so much inaccurate as already somewhat dated. Zimmer could not have predicted COVID-19...

...reports where someone tries to “optimize” a particular standard lab protocol. For example, someone may attempt...often have to start pursuing other options. While some non-academic career options require additional study... answer loads of questions, and I also had to do some troubleshooting as advised by the technical support...required for a role in technical support? It takes some time (several years) to build up comprehensive lab...companies have dedicated technical support teams, sometimes comprised of several layers of support: A front-line...often lead to otherwise unobtainable solutions. To some extent there is the need (or opportunity) to get...listen to the many individual stories and get (sometimes refreshing) insights into how other scientists...

...s natural that some of us have more in-depth knowledge of one area, and when something urgent comes up... things in our code. In some parts of our code, we might be calling something a sample and in other places.... We support everyone that works with Addgene in some way. For users, we just released a major redesign...genders, educational, and professional backgrounds. Sometimes this can lead to wildly different ideas of how...during checkout. Demo details for this would be something along the lines of showing that the checkout takes... decide how to best approach it in our code.  Sometimes while thinking through these steps, we discover...fixing bugs: we take time during each sprint to do some maintenance work on our code. One way to look at...

...also require some additional optimization as each antibody performs differently and some antibodies impact...of interest within an intact cell, tissue, or, in some cases, a whole organism. But, as with any antibody-based...are essential. In this article, we’ll go through some of the most important considerations to make when...see fluorescence indicates your target’s location. Some other terms you might see associated with IF are...visualize the antibodies using fluorescence (for some background on fluorescence, try the Introduction...straightforward than choosing a primary, so we’ll start there. Some of the most important considerations are to make...applications, including IF (or IHC or ICC), and will share some of their results and corresponding protocols. Other...

...resistance cassette. This leaves a “scar” in the chromosome, sometimes interfering with expression of surrounding...There’s an astounding number of ways to create chromosomal mutations in bacteria, so many that it may be...quick and easy way to introduce a mutation in the chromosome is to disrupt expression of a gene with an antibiotic...ones that don’t leave any undesired scars in the chromosome. These methods roughly fall into two categories...introduced from a conjugative plasmid replaces the chromosomal copy. This is a versatile technique, and is less...large amount of user-to-user variability, and in some cases, counter-selection escape can occur: single...inserted a copy of rhaS with a strong synthetic ribosome binding site (using the online calculator from...

...First, you’ll need to find someone who is willing to host you! If there is someone you personally know or ...scientist? A visiting scientist, in academia, is someone who visits an institution to perform research there...is because the host lab or institute provides something that you can’t obtain at your current institute...research needs, that is probably the easiest path. Some institutes have structured visiting positions within... These structured types of placements often have some form of built-in professional development. These...be right or necessary for many scientists! Unless some of these logistics are covered by a structured visiting...notes, and ask questions if you don’t understand something. Offer to help out wherever you can and don’t ...

...will give you some advantage over other candidates. Write about your transferable skills Some of the skills...'t think a resume is "one size fits all" like sometimes CVs are. Because you only have two pages, you ...collaboration between research groups? Perhaps you had some supervisory experience working with undergraduate...students? On top of that, perhaps you were involved in some extracurricular activities – did you volunteer to... about science? Or perhaps you have presented in some public lectures? These transferable skills could...think only undergraduates use these services, but some of the general knowledge about preparing a good ... dealing with interviews, is actually universal. Some universities even have officers working specifically...

...restriction digests on plasmids at Addgene, we sometimes observe something odd in our uncut DNA control: a band ...MiSeq sequencing that Addgene uses.) However, when some researchers received the sequencing results for ...form of the plasmid with repeating sequences. In some specific cases, multimerization has been observed...please note that the supercoiled dimer band can sometimes overlap with the band corresponding to the nicked..., it cannot be used to detect plasmid multimers. Some companies provide NGS whole plasmid sequencing services... reduce the frequency of multimerization, and in some cases completely prevent the formation of plasmid...Screen multiple colonies to isolate the monomer Sometimes a plasmid can exist in a bacterial population ...

...full-time position. For my part, I really enjoyed some aspects of the work, such as physically carrying...failure and problem solving. I believe that is something lacking during degree courses; they never really...the nursing students and community health workers some basic biology and first aid. This really allowed...the company was to fulfill contracts, my work sometimes felt rushed and the focus always seemed more on...skills in industry positions. There were, however, some downsides to working in a large commercial company...situation, communication between departments was sometimes difficult and the solution attempted was more ...solved with good management practices, but can sometimes go unnoticed at large organizations. Another downside...

...mental effort, and that some concepts are easier to grasp than others. But sometimes we’re unaware that we... a visual aid instead of describing something verbally. Sometimes a group was just really tired and needed...as it does to read an 11-page Cell paper - and sometimes, it might actually take me less time to read the...contributes to cognitive load?  Everything! Here are some common areas that can use up your audience’s mental...encouraged) to leave blank spaces or to gloss over some aspects when needed. The trick is to communicate...way I thought about engaging with learners. If something I was communicating didn’t click, instead of blaming...put their phones away), or maybe I was assuming something was intuitive that actually wasn't. Maybe I needed...

...viruses that can infect people, cells, and animals. Some of these viruses are completely inert, but others...controls, safety systems that are built into how something functions, were designed for this purpose. One...understand what safety precautions to take, and if something does happen, ensure you can receive appropriate...monitoring. Virus-specific concerns   In nature, some viruses are more dangerous than others, and the ...infection process (though it can integrate at chromosome 19q13.4 qtr. (AAVS1)). For these reasons, AAV...does not integrate into the host’s genome. However, some adenovirus strains are known to cause respiratory...peracetic acid, 10% bleach, or 1% iodine are useful. Some organizations or cities may require a higher percentage...

...Professor Emeritus at UCSF. Dr. Bourne has some radical (and some not so radical) suggestions for defining...worked with some great postdoc and grad student career offices in the last few years. However, some organizations...scientists can have successful careers doing science in some form? There is a lot of talk about the state of ...rumors that we are training too many scientists and some propose radical changes to the way we view the graduate...organizations are woefully behind and some just don't have the resources to do enough. There are still university...in taking matters into their own hands, there are some easy ways to get started. I am happy to work with...

...review the inspiration behind ExM, how it works, and some of the limitations to keep in mind whether you are...option, but the equipment needed for EM is not something you find in the average cell and molecular biology...digest the sample to allow for even expansion.  In some cases, labels are added after this stage.  Step...may be better for your specific targets. What are some of the limitations?  A “super-resolution” technique...sounds great, right? But there is always a catch - some limitation of the technique that users should be... continuous lines in pre-expanded samples can sometimes take on a fragmented appearance post-expansion...can be alleviated by maximizing label density. In some cases, nanobodies have been found to help improve...