... takes an hour or less to complete at 50 °C. After an hour or so, the sample is immediately ready to transform...to design primers for Gibson Assembly. For a simple example of using Gibson assembly, imagine that you ...will go over some advantages, disadvantages, and examples of how scientists are using Gibson assembly to...assembly is well known for allowing easy assembly of multiple linear DNA fragments, but can also be used in ...biology, mainly because of the ease in assembling multiple fragments in one step with no scar sequences remaining...where no other methods were available. A second example of using Gibson Assembly along with CRISPR was ...Whether it is for routine cloning, assembly of multiple fragments, or synthetic biology, you should consider...

...platform that allows us to provide scientists with complete plasmid sequences. After launching our viral service...accommodate adeno-associated viral vector (AAV) samples and created a simplified process, termed viral ...parallel, there is a very real risk of mixing up samples, serotypes, or both. Unfortunately, the standard...present SMRT sequencing only works for self-complementary AAVs and the TA-based ligation and tagmentation...molecules in the DNA extract associate with complementary strands, enough double-stranded species exist...Pacouret, 2017). Moreover, the sequencing data from multiple AAV lots can be run in parallel and provide serotype...adeno-associated virus particles containing complementary polynucleotide chains. J Virol 9(2): 394-396...

...hand. Records of plasmid construction were often incomplete or nonexistent. In the 21st century, many molecular...molecular biologists didn’t know the complete sequences or properties of the DNA molecules they were using... (HCI), and we have adhered rigorously to HCI principles. For every task, we envision what the user wants...refinements in response to customer feedback. An example of this approach is SnapGene’s algorithm for detecting...could annotate a feature inaccurately. The best example is fluorescent proteins, which often come in closely... harness common feature detection in a new way, please contact us at snapgene.com. Many thanks to our... She is passionate about building software that people love to use, and likes to start at the beginning...

...shown in the image below. An example of Gram positive (purple) and Gram negative (red/pink) staining...the CV+, creating a complex within the peptidoglycan layer, staining the cell purple. When the alcohol ...thinner membrane. Procedure The stain is a pretty simple process. First, flood the heat-fixed smear with... Gram positive bacteria will be stained blue or purple, and gram negative bacteria will be stained red...the peptidoglycan layer, trapping the large dye complex created by the interaction of CV+ and I- in the...staining procedure, gram positive bacteria are stained purple or blue, and gram negative bacteria are stained...

...need to give everyone a heads up (for example, notifying people of an updated protocol), send an email...Google doc where people can make comments. At meetings, we just focus on the parts where people had questions...steps (see “action items” below). Get the right people in the room There are few experiences more frustrating...our all-company meetings short, informative, and pleasant. Email: Sometimes, the right meeting is no meeting... positive peer pressure to make sure everyone completes their assigned items to keep the project moving...happen “off-line” with just one or two relevant people. How often do you need to meet? Use trial and...

...The first couples NLuc to fluorescent proteins to make better reporters and the second couples NLuc to ...are available from the repository. We encourage people to go to Addgene to get new innovative tools. Afterall... NanoLuc® Luciferase-based tools are fantastic examples of technologies that can accelerate your research...endogenous protein tagging as it is only 19 kDa. An example is the CRISPaint-NLuc construct (Plasmid #67178...non-optimal for in vivo imaging. The solution is to couple Nluc with a red-shifted fluor. Here, NLuc internally... Nano-Lanterns  Suzuki, K., et al. (5) imaging multiple proteins at the same time in live cells using ... but has been hampered by dim signals from the coupled luciferase. This strategy was first applied using...

...experience. I enjoyed helping people and learning to explain concepts in ways that people from another background...This was particularly interesting as I had just completed my thesis on the study of e2Fb, which is a tumour...cancer and I loved the practical aspects of testing samples in the lab, but I really struggled with the large...strong point. Working in industry: virology On completing my MSc, I again had to look at what type of work...English speaking country with a strong economy and plenty of jobs in science at world renowned institutes...loved the fact that my work had a real impact  people’s lives and would improve their treatment outcomes...of a dream job, but I knew that I enjoy helping people and decided to compromise and applied anyway. I...

...𝛽-galactosidase. Complementing this mutation by expressing this deleted portion, for example on a plasmid,...fewer colonies after your experiments. As a common example, a selection will leave you with the colonies that...plasmid contains your insert of interest or not. For example, choose enzymes that only cut once on either side...you will just see one band. These are only a few examples of how you can use restriction digest to screen...get started, you will first need a primer that complements your plasmid sequence. Start with a backbone-...backbone-specific primer that will sequence over the Multiple Cloning Site (MCS) and into your insert. Check ...can confirm the sequence of the entire plasmid. Please note that NGS verification is more time-intensive...

...scientists around the world. In 2004, that was a fairly simple goal… and we had no idea how much our understanding...text into our work, we began to realize how the principles we used in our daily content writing applied ...writing in scientific writing, and how often the principles we developed to use in our website and educational...Steps to clear writing Let’s use Figure 1 as our example. Remember, our goal is to use words to describe...Gradient Ultracentrifugation. The reader may be, for example, a graduate student using viral vectors for the...process, try writing your own alt text for the example image. We’ll wait!  Done? Excellent! We asked our...and help make your paper accessible to even more people.  Whether you start small with alt text or immediately...

... synthetic transcription activation complex consisting of multiple distinct effector domains that work...well enough on any gene that they could be used by people with little bioengineering expertise? “This is ... Mediator or SAM. SAM is an engineered protein complex for the transcriptional activation of endogenous...with a BRAF inhibitor. (The test was the perfect complement for one conducted earlier by the Zhang lab for... These libraries are lentiviral in nature, so please ensure that you are equipped and authorized to ...sam.genome-engineering.org/. Crystal structure of Cas9 in complex with guide RNA and target DNA. Nishimasu H, Ran...transcriptional activation by an engineered CRISPR-Cas9 complex. Konermann S, Brigham MD, Trevino AE, Joung J, ...

...Senior people (of which there are never enough, especially in underrepresented groups) reach multiple mentees...share a similar direction or problem. For example, a group of people who are all interested in making a transition... at my company, but there weren't enough senior people willing to step up and be matched with the many... or both of these criteria. If you can, include people from different labs, departments or organizations... such as pre-reads, videos or exercises to be completed in preparation for the meeting or to be read or...event together and help each other practice meeting people – compete for fastest to set up a coffee date or...

...interested in expressing multiple small genes, you can design single rAAV vectors with multiple genes packaged ...indicate the ITR and serotype, respectively. For example, AAV2/8 has a type 2 ITR and a type 8 serotype....interested in heftier genes, you can coinfect with multiple viruses to express genes or gene sets over 4.7...conventional single-stranded AAV (ssAAV) and a self-complementary AAV (scAAV) vector. Full-length ssAAV vector...2.5Kb) and you need expression in a hurry, self-complementary AAV (scAAV) may be an option (McCarty et al.... E. Monahan, and Richard J. Samulski. "Self-complementary recombinant adeno-associated virus (scAAV) vectors... Multiple Authors...

...presentation, lost in a sea of data, my mind went completely blank. After several seconds of truly panicked...honest, if my paper hadn’t been ridiculously more complex than the one before it, I don’t think I would have...blocks. The problem is, when it happens to other people, it’s normal and quickly forgotten, and when it...and awful. So try to keep some perspective. Most people won't notice, or won't remember and those who do...dumpster noises were just last week!  Nerves It’s completely normal to be anxious, nervous, and/or apprehensive...still get nervous quite regularly, and I've had multiple jobs where all I did was public speaking. If you're...make a strategy that can help. Even something as simple as telling yourself, “I can stop and check my notes...

...silencing complexes (RISCs), which detect dsRNA in the cytoplasm and use it as a guide to complementary RNA ... DNA at risk. Consequences of duplexed RNA Mammalian cells have multiple receptors in place to sense dsRNA... pathogenic proteins, as well as in the lab to deplete expression of proteins of interest. RNAi has even... cytosol. Fig. 2: Endogenous sources of duplexed RNA DNA:RNA hybrids Sometimes ssRNA just needs...intermediates in the process. These transcription-coupled R-loops are transient in nature and will dissociate...dissociate when gene transcription is complete. However, transcription of thousands of genes is constantly...

...QCascade complex surveys the genome and binds its target DNA sequence. The transposase complex binds the...really depends on where a transposon lands. For example, in bacteria, transposons can move antibiotic resistance...is left behind. When the transposon-transposase complex finds its target site, it integrates, which produces...TEs You can think of autonomous transposons as “complete transposons” since they encode the protein they...selecting a transposon for a mutagenesis screen. For example, piggyBac and Tol2 are best for screening promoters...DNA-targeting module with the crRNA, 3) a transposase complex made up by three transposase proteins (TnsA, TnsB...

...ligands to the same sample to label sub-populations of your protein of interest. For example, the Cohen lab...derivatives of its substrate O6-benzylguanine (BG) (Keppler et al., 2003). This engineered hAGT later became...multi-component systems can be complicated to implement, they can be highly specific with a small and ...Atto590-BG (green), imaged with stimulated emission depletion (STED) nanoscopy. Scale bar, 2 μm. Inset shows...proteins in vitro, you can even use the same tag for multiple proteins but label each separately before reconstituting... assembly-regulating proteins in vitro reveals complex events. a) Micrographs of a fluorescently-labeled..., biotin, or magnetic beads. In one innovative example, the Zhang lab used SNAP-tag to decorate synthetic...

...when studying them since their complete knockdown is lethal. Incomplete or titratable knockdown, however...the tools scientists can use to study them. For example, gene knockdown with CRISPR interference (CRISPRi...wall. Once in the recipient cell, the CRISPRi DNA (purple) flanked by left and right Tn7 end sequences (green...showed that Mobile-CRISPRi is transferable into multiple bacterial species including several involved in...pathogenic strains and environmental isolates. For example, Peters and colleagues used Mobile-CRISPRi to knockdown...if that gene is a target of an antibiotic. For example, let’s take a look at the essential gene folA which...also possible with Mobile-CRISPRi. As a proof of principle, Peters and colleagues constructed a 40-member...

...increases. Multiple inserts: There are some cloning methods that make working with multiple inserts a ...as some methods are more amenable to combining multiple DNA fragments than others. In today’s post, we...with a special emphasis on these five categories. Please note that many of the following cloning methodologies...interested in learning more about any one of them, please click on the links throughout the text or see the...per reaction but can be used to quickly generate multiple clones. Difficulty: If you’re an experienced cloner...Golden Gate assembly allows one to simultaneously complete an ordered set of scarless ligations following...one right answer, so don’t be afraid to explore multiple approaches to find out which one works best for...

...into multiple cell types of the same lineage. Blood (hematopoietic) stem cells are a good example of multipotent...mostly to maintain cell numbers or replenish dying cells. For example, hematopoietic stem cells found in...functions [1]. In many tissues, stem cells serve to replenish/replace damaged cells that no longer function ...[1]. Stem cells’ ability to differentiate into multiple cell types makes them useful models for developmental...thought of as descendants of totipotent cells. An example of pluripotent cells is embryonic stem cells (ESCs...in science communication and publishing after completing her postdoctoral training from Massachusetts ...

...yield. Keep in mind that you might need to use multiple techniques for a particularly stubborn prep. ...remove endonucleases (see figure at right for an example). Look out for carbohydrates Certain strains, including...the cell lysis process. To ensure that lysis is complete, use a reagent like Qiagen’s LyseBlue, which uses... uses color to show you if lysis is incomplete. Protein expression strains are not good for cloning Bacterial...yields. Why would volume make a difference? In the simplest of terms, more volume = more cells = more plasmid...culture media Changing to a richer media or adding supplements can improve your cell density, leading to an ...normally recommended antibiotic concentration. Add supplements to the media You can achieve higher cell density...