...repeats separated by an 8-bp long asymmetric core spacer sequence. The asymmetry in the core sequence gives... double-strand DNA break occurs within the core spacer of each loxP site and the two strands are ligated...loxP sites is reversed.   Deletion: If the sites face in the same direction, the sequence between the ...been created, which contain a unique asymmetric spacer "NNNTANNN", where "N" indicates which bases may...

...edit. Figure 2: Cytidine deamination takes place on the free strand of DNA and converts a C to U ..., ABE8s resulted in 1.5-fold more editing at protospacer positions A5-A7 and 3.2-fold more editing at ...Joung’s lab created a dual-deaminase editor called SPACE (synchronous programmable adenine and cytosine editor...mechanism than base editing, has allowed scientists to replace entire sequences of varying lengths. Base editing...and adenine base editors Grünewald et al., 2020 SPACE   Fused cytosine and adenine base editors Walton...

...PubMed search option in early 2020 and eventually replace the existing version of PubMed.  Key features This...references sections of a PubMed entry are great places to look for more articles on your topic of interest...Twitter to promote their work, so it’s also a good place to look for publications. Bonus: papers have to ...automatically update as papers are published, similar to Facebook or Instagram. Key features Setting up a personalized...

...structures, etc., as well as the presence of a Protospacer Adjacent Motif (PAM) sequence. If the gRNA sequence...extent NYN. This increases the flexibility of gRNA placement significantly over SpCas9 and more so than Cas9s...Engineered CRISPR-Cas9 nuclease with expanded targeting space. Science 361,1259-1262(2018). DOI:10.1126/science.aas9129...

..., if these variants are fused to the MLV Gag in place of SpCas9. gRNA A gRNA targeting a DNA sequence ... charge repulsion between virions and the cell surface, and also helps form DNA and retroviral-particle...components. However, this sometimes requires intracellular injections into the cytoplasm or pronucleus ...avoid this pitfall since they are injected into the space surrounding the zygote, where they can then fuse...

...contained all 64 possible NNN PAM sequences adjacent to a protospacer. If ω-dCas9 could recognize a library ...other CRISPR enzymes require a short PAM sequence adjacent to the targeted sequence at the locus of interest... and 3.7 shows that we have only scratched the surface of modifications that can be made to Cas9. It is...

...ultrafugation Overview: Iodixanol gradient ultracentrifugation uses a gradient of different concentrations...during AAV production is overlaid. Following ultracentrifugation, the AAV-containing 40% fraction is collected...day. Refer to the AAV Iodixanol Gradient Ultracentrifugation Protocol for more details. Pro tips Watch...transfer plasmids. It’s easy to contaminate a workspace with plasmid, not that I would know about this...

...quite large (500-600 bp) and may take up precious space in viral transfer vectors with limited packaging...stream of the IRES or 2A element (depending on the placement of the reporter gene). Addgene's collection offers...flanking the multicistronic element(s), or you could replace one of the reporter genes above with your gene ... are designed to have one or more of the genes replaced.  Additionally, 2A peptides could be PCR-cloned...

...gene therapy had fallen - plummeted really -  from grace 2 years prior with the death of Jesse Gelsinger ...these key elements together at the right time and place. But it is the hard work and persistence of a dedicated...early as next year. Twenty years after falling from grace, gene therapy is back, better and safer. It is now...therapy for blindness 2. Gene Therapy emerges from disgrace to be the next big thing, again 3. Acland G. et...

..., the real value here is seeing them all in one place. Can we address problems in scientific publishing...of creating a technology that could eventually replace scientists themselves.  The sections on optogenetics...COVID times,” but the pandemic has certainly exacerbated them and forced scientists to change their priorities...introduction they need to grasp the challenges you face in your career.  Acknowledgements: I would like...

...is then washed, and either eluted from a fixed surface or precipitated, dried, and then rehydrated. There...glass particles in glass milk, gel slices are first placed in a chaotropic salt sodium iodine (NaI) solution...with ddH2O and then ethanol. After regeneration, trace amounts of DNA (3 femtograms/ul) could still be ...cleaned with 1M phosphoric acid and re-used, but trace contamination could interfere with interpreting ...

...yellow derivatives) A206K Interferes with dimer interface (monomeric derivatives) K26R, Q80R, N146H, H231L...biosensors has been designed to detect a variety of intracellular conditions, including ion (such as Ca2+) concentrations...available. Others: We’ve really just scratched the surface of the potential applications for GFP. It has also...strand break repair, and to label pathogenic intracellular microbes to visualize host/pathogen interactions...

...express my concerns and request a replacement. I got my replacement in the end and the issue was resolved...less suitable for troubleshooting or issuing replacement products. In order to find good and helpful answers... in you – a technical support role is the right place to live it out, but ideally without asking too many...

...progress. You will also probably have a lot more face time with your bench mentor than your scientific...lot of details, especially when you are in a new place, learning something new. So how can you remember... walking through the protocol, by going to each space in order to double-check you remember where the ...Resources Resources on the Addgene blog Trading Spaces – Visiting Scientist Edition Mentoring for Scientists...

...target is present immediately upstream of a Protospacer Adjacent Motif (PAM), the sequence NGG.  If these... within the editing window (bases 4-8 of the protospacer). While this is a small window, this system can...system.  Fig. 1: Cytidine deamination takes place on the free strand of DNA and converts a C to U ...

...recombination; SSA, single strand annealing) took place and reconstituted the EGFP expression cassette. ...fragment containing the sgRNA target sequence can be placed in multi-cloning site (MCS) between EGFP fragments...extra precautions needed when dealing with RNA (e.g. face mask, and a dust free and specially cleaned work...mice that may have no mutation, costing money and space. As a rule, screen all of your pups unless you have...

...), the inner membrane space of mitochondria and chloroplasts, the extracellular milieu, and gram negative...should be taken into account when deciding where to place an FP within a fusion construct [1,2], i.e. positioning...mitochondria, lysosomes or they are secreted into the extracellular milieu. Many of the resident proteins within...

...is by no means exhaustive, but should be a good place to start when trying to pick your perfect promoter...promoter down regulation due to methylation or deacetylation. Ubc General expression mRNA Mammalian promoter...calmodulin. GAL1, 10 General expression mRNA Yeast adjacent, divergently transcribed promoters Inducible with...orientation to your gene Although this list is a great place to start, the tables above do not delve into the...

...The source of those conflicts can frequently be traced back to one of a few trouble spots:  Communication...here is pretty intuitive: You need to be willing to face conflict head-on. That doesn’t mean you have to ...and compromising. Though there may be a time and place for avoiding, competing, or accommodating, if you...knowledge to resolve conflicts. Conflict in the workplace is inevitable, but having who can help resolve...

...consuming and difficult. Finding volunteers who had space to shoot was another hurdle. As we slowly started...reopened. But there were still many restrictions in place, most importantly, masks.  Besides the muffled voices...strict safety protocols to keep Addgenies safe. Space was at a premium, so I had to figure out a solution... we were finally refining the process down to a place I could be happy with it. Cycle 5 - The Future! ...