...revolutionised the way we apprehend molecular complexes and is still a very useful tool.    Like a guitar...technique, but it is very important to follow some simple rules to avoid common pitfalls and to use it at... These issues can be overcome by choosing a complementary pair of fluorophores. To maximize the FRET signal...especially when you investigate unknown molecular complexes. But it is a parameter that you have to keep in...most efficient approach for your own system. The simplest and the most popular one is the sensitised emission...photobleaching method The acceptor photobleaching method is simple but limited to a single measurement. This method...

...endogenous homologous recombination enzymes can also accomplish the desired genomic modification. This technique... which results in a merodiploid. This can be accomplished by selecting for an antibiotic resistance marker...Valentovich 2014). We routinely clone the purified amplicons upstream and downstream of the desired deletion...included versions with gentamicin-resistance (amplified from TP997). We validated these vectors, which...scarless modification of bacterial genomes. It can accomplish large deletions, insertions, and even precise...system with lambda red recombineering enables simplified chromosomal gene replacement in Escherichia coli...

...locations using Cas9 and track the localization of the complex. Broccoli, the other green fluorescent aptamer,... RNA. Fluorescence is observed when these RNAs complex and the entire aptamer can hybridize and fold. ...uniqueness of the photostability of the Corn-DFHO complex. For dessert (last but not least), the only fruit-named...Other groups have used Mango to study RNA-protein complexes (Panchapakesan et al., 2017) and short non-coding...mango aptamer-fluorophore: a bright, high-affinity complex for RNA labeling and tracking." ACS chemical biology...transcription using a photostable RNA–fluorophore complex." Nature chemical biology 13.11 (2017): 1187. PubMed...

...tough. Is the content accurate? Are we conveying complex ideas in the easiest way possible? Are the shots...Magic Pocket Cinema Camera 4K. It’s wonderful and simple to use, but when you need to juggle filming and...perfect height (Fig. 2). We recorded video using a simple, external video recorder and a mic clipped to her...video conferencing software. Interviewees could simply boot up Zoom and record themselves talking to their... and availability, a traditional solution was completely out of reach for Addgene.  So, onto research!...  Figure 4: The remote directing setup, complete with wireless video receiver, camera control, ...

...multiple plasmids. For simplicity, we’ll use the second-generation system as an example.  The lentiviral ...double-stranded DNA transgene is incorporated into a large complex of both viral and cellular proteins and trafficked...the delivered payload is gradually reduced. For example, after using CRISPR/Cas9 to edit a gene, the expression...integrated piece to measure! Instead, two commonly employed methods for titering IDLVs include detecting vector...performing quantitative PCR titration of vector DNA in samples harvested 24 hours post-transduction (Wanisch and...

...CRISPR may be the way to accomplish this tough job! Kamel Khalili’s lab at Temple University has demonstrated...the T-cell line 2D10. PCR amplification and Sanger sequencing of pooled samples showed that most of the ...undetectable in the blood. But the virus doesn’t completely disappear; it just hides in latently infected...the immune response. The second strategy is the simple removal of the HIV-1 genome from infected cells...

...postdocs. Unfortunately, even when universities have simple postdoc numbers sorted out there can be gaps in...Postdoc: An Institutional Perspective. This lack of complete information does not, however, prevent us from...scientifically minded, and able to communicate complex information - all traits you are likely to find...important take-away from this session was the idea that simply satisfying a percent requirement for diversity ...difficult question but not one that should be abandoned simply because it is difficult and is key to the continued...research. Panelist Moon Duchin provided a concrete example of how underrepresented individuals are discluded...

...oligodeoxynucleotide HDR template covalently attached to the Cas9-gRNA RNP complex via the fused HUH endonuclease...genome-wide CRISPR inhibition Covalent tethering of DNA template to Cas9 SECURE base editors Nme2Cas9 CRISPR interference...inhibition library Covalent tethering of DNA repair template to Cas9 Homology-directed repair (HDR) can be ...Neisseria meningitidis, Nme2Cas9, that recognizes a simple dinucleotide PAM (N4CC), lending to high target...

...results Above (top right corner of this post) is an example of a seemingly clean DNA sequence (no Ns in sight...less and you might suspect contamination in your sample or consider asking your sequencing facility to ...to apply a special protocol for a difficult template. Anything more and you’re venturing into the uncertain...Use a silica spin column for purification of the samples you send for DNA sequencing If your sequencing ...requires you to perform your own Big Dye PCR amplification reaction (as opposed to using the all inclusive...

...This was particularly interesting as I had just completed my thesis on the study of e2Fb, which is a tumour...each advertised job received many applications, employers were requesting that only scientists with an MSc...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... solution attempted was more meetings, but this simply seemed to create more bureaucracy. As with any ...to a Work-Travel VISA, which means I can gain employment for up to 6 months in Australia or another country...

... to an internship on the journey to full-time employment. I have always been interested in the ways that...the forefront, no longer allowing myself to just simply exist without creating a storm. Given all the options...Canva, and I was able to create cute and on-brand templates for these posts. Some of recent Meet an ...what it was like to work at Addgene. This is exemplified in my work interviewing fellow Addgenies. I spoke...company that cares - not only in terms of its employees but in general about anyone who steps foot through...

...from at least some employees. Watch out for "information allies" – these are employees in your group that...feel that giving feedback will be perceived as complaining. They may worry that it seems like "tattling"...have understood the speaker accurately. Thank employees who volunteer constructive feedback and suggestions...influenced by feedback. If you don't do this, employees will stop providing feedback pretty quickly. ...

...recombination that are typically employed during viral plasmid amplification (Fischer, KB. 2019). This means...expression profile at multiple steps from plasmid amplification in bacteria to transgene expression in vivo....of spontaneous reversion during AAV plasmid amplification. On the left homologous pairs of scrambled cre-recombinase... set of injections into your animal model. For example, in a recent study by the Wickersham lab (Lavin...essential that every new vector, whether it be a completely new transgene or merely a new lot, be thoroughly...

...this biological lesson and created a system to amplify fluorescent signals. Named for the "stellar explosion... However, this system is far from perfect. For example, If your fusion protein is expressed at low levels...expressed from a separate plasmid. This system amplifies the intensity of the fluorescent signal and enables...chance of phototoxicity Reduced photobleaching Simplified, long-term single-molecule tracking Caveats ...et al. “A protein-tagging system for signal amplification in gene expression and fluorescence imaging....

...this can be accomplished with either a PAM-in or PAM-out orientation. Just as the names imply, PAM-out designs...Overview of Cas9 nickase We’ll use SpCas9 nickases as examples for this post. The D10A mutation inactivates the...site rather than the optimal <10 nt distance. Employing two gRNA sites that are further away from the ...mutation site. Quick tips for nickase design Ready to employ nickases for your next CRISPR experiments? Here...

...medium containing the corresponding antibiotic. For example, if the DNA you’re inserting into the plasmid backbone...an essential amino acid), and a plasmid that complements the auxotrophy (i.e., synthesizes the critical... resistance spontaneously arises in yeast. For example, Markus Ralser’s lab created a yeast prototrophy...prototrophy kit containing 23 plasmids to complement auxotrophy. Negative selection: Cells that have lost a specific... gene. SacB can be used similarly to CcdB. For example, in the gene expression plasmid pGTvL1-SGC from...

...resolution as receptors can be turned on and off simply with the presence or absence of the correct wavelength...increasingly faster kinetics. The opsin ChETA, for example, is a ChR2 opsin mutated to have faster off kinetics...studies, however, require a permanent intracranial implant to be surgically installed to allow for light delivery...flexible as it relies on designer ligands that can be simply introduced by injection. The main challenge in ...Invasiveness Mice require permanent intracranial implant Noninvasive Controlling Stimulation ExogenousEasily...

... last step will influence the stability of your sample over time and determine which experiments you can...most common E. coli strains used for plasmid amplification are endA negative, meaning the gene has been... impact the time you will be able to store your sample without having substantial degradation and which... DNA, it will limit what you can do with these samples. This is because divalent cations are essential...reaction, an effective possibility is to dilute your sample in water to reduce the EDTA concentration. Alternatively...

...response to a viral infection a) antibody-dependent complement deposition (ADCD) b) antibody-dependent cellular... Erp, et al., 2019.    Antibody-dependent complement deposition (ADCD) Antibodies can bind to a pathogen... known as opsonization. In antibody-dependent complement deposition (ADCD), opsonization of IgG and IgM...pathogen activates the immune system's classical complement pathway, starting a cascade of events that results.... PMID: 10742152. Dunkelberger, J., Song, WC. Complement and its role in innate and adaptive immune responses...

... models. In complex diseases, it can aid in generation of multiple edits or implementation of expression...change in pathology. If the disease pathology is complex, a model may be most effective. Once drug candidates... all diseases are fully characterized or have a simple pathology. In these cases, preliminary identification...made this control step extremely accessible. For example, let’s say you identified gene target X for BRCA-deficient...