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... the fragments size of your digestion on an agarose gel. If the digestion does not provide the fragments... to your cloning strategy: Restriction enzyme digestion Be mindful of the restriction enzymes you chose...like XbaI, ClaI, etc. If you are doing multiple digestion be sure that the buffers and temperature are compatible...It's also best to use 1-2 ug of the vector for digestion. PCR Design primers that overlap at least 24 bp...

...cytosine and adenine base editors that can be reconstituted in vivo to form a protein identical in sequence... plasmids and co-transfected into cells for reconstitution. Image from Levy et al., 2020 with permission...when paired with appropriate fluorescent optics. Existing red fluorescent voltage indicators, however, fall...neural circuit manipulations when combined with existing green-fluorescent GEVIs and blue-light-sensitive...The kit functions in the model cyanobacterium Synechocystis sp. PCC6803, and the fast growing strain Synchococcus...

...available to counteract the toxin. If, however, an investigator were to successfully clone an insert into the... (re-ligated empty vectors, for instance) would still express ccdB and therefore would die. This procedure...insertion will completely replace ccdB with the investigator’s insert of interest. Hence correct clones are... behind the “swapping out” of ccdB with the investigator’s gene of interest.   CcdB resistant E. ...further. Feel free to offer your comments and suggestions, and even to submit a guest blog entry of your...

...controlled by deliberate and compulsory actions instigated by the central nervous system signaling to peripheral...metabolic syndrome. Coregulators Coregulators constitute a group of some 400 molecules that have been ...regulated manner. The fact that coregulators belong to distinct complexes may explain how more than 300 different...collectively address many important and fundamental questions in nuclear receptor and coregulator research. ...the NURSA website will feature direct links to listings of available plasmids for our curated NRs and ...

...penicillin resistance plasmid can grow in spite of a hostile Penicillium fungus trying to kill them off. While...resistance to toxic substances like heavy metals or pesticides, enabling their hosts to survive where others... This ability to endure a harsh environment is distinct from the benefit of resistance plasmids, which...shifted over the years, and new classifications are still being created as whole-genome sequencing becomes...Plasmids Isolated from Lactic Acid Bacteria:Their Characteristics and Potential Applications in Biotechnology...

...how close is close enough for the desired edit to still occur if the cut site is sub-optimal? As with everything... cut site is just 10 bps away from the site in question (Kwart et al, 2017). To add insult to injury, ...same PAM preference of NG was isolated by the Kleinstiver Lab (read more on this lab's other developments...Nonetheless, in an AT-rich region, these Cas9s may still leave you choosing between efficiency and location...location.   Nearly PAM-less editing   The Kleinstiver lab sought to address the PAM problem through structure-guided...

...ball-and-chain that they resemble, actually impede your investigation into the regulation of your favorite gene. But...these studies report correlations without clearly testing whether a certain methylation state causes a condition...to create rare DNA bases that are recognized by distinct proteins and result in unclear transcriptional...achieved using lentiviral backbones expressing blasticidin or puromycin resistance genes.  It’s that simple...Feinberg A, Vogelstein B (1983) Hypomethylation distinguishes genes of some human cancers from their normal...

...certain cell types.  Botond Roska’s lab at the Institute of Molecular and Clinical Ophthalmology Basel ...finding retinal cell-type specific promoters. Most existing promoters express in a broad number of cell types...targeting the same cell class in all three species are still greater than random chance. The chances of a promoter...human retinas but not in mice was low (R=0.12), suggesting that mice could be used to eliminate promoters...Morikawa, R.K., Rompani, S.B., Hantz, P., Szikra, T., Esposti, F., Cowan, C.S., Bharioke, A., Patino-Alvarez,...

...a risk that they may underestimate viral titer as these assays cannot distinguish between a cell that ... based off of p24 quantification tend to be overestimates since they can include free p24 and defective...assay, titering via measurement of viral RNA can overestimate the amount of infectious virus in a prep as ...integration events, reducing the possibility of underestimating the viral titer. The main drawback of this ...

...engineered precursor of the systemic capsid in question (or both). Therefore, a high-efficiency systemic...These are only starting recommendations; you will still need to experimentally determine the best titer ...your experiment, it’s time to start selecting and testing systemic capsids! Figure 2 contains a decision ...reduced efficiency, and is not neutralized by pre-existing antibodies against AAV9-based vectors (and vice...AAV-PHP.S, with a bias toward segments of the small intestine (Chen et al., 2022). Systemic capsid for alveolar...Cre-dependent selection yields systemic AAVs for targeting distinct brain cell types. Nature Methods, 17(5), 541–550...

...protocol for your pooled library deposit. When a requesting scientist receives a pooled library from Addgene...colonies, you’ll likely rely on serial dilutions to estimate transformation efficiency (Figure 2).    ... Figure 2: Example of a serial dilution to estimate transformation efficiency. Figure used with permission...protocol paper by the Zhang lab, and the Broad Institute’s general pDNA Library Amplification protocol are...depositing it with Addgene. If you have any specific questions about designing your library amplification protocol...

...the prep during the production process? These questions are especially important when producing multiple...we have analyzed hundreds of viral vectors and estimate that recombinase-independent recombination occurs...below to let us know if you have any specific questions about viral DNA NGS you’d like us to discuss. ...by phone or at [email protected] to answer your questions about our quality control process! The images...Vector Characterization Methods for Quality Control Testing of Recombinant Adeno-Associated Viruses. In: Methods...

...in 5’ four base overhangs after cutting. The destination vector receives the assembly after similar Type...re-digested and "eligible” for ligation into the destination construct. Reactions are often cycled between...acquisition of antibiotic selectable markers by the destination plasmid. Antibiotic selection allowed for high...very occasional uncut or cut/re-ligated pGGA destination plasmids. A final validation of the 5-, 12- or...is dependent on the presence of every module, destination construct and functioning Type IIS restriction...

...discuss the work, set expectations, answer any questions, and (most importantly) decide on a timeline. ...regularly, make yourself available, and encourage questions. Unless you’re delegating a recurring task to ...to a seasoned report, there should be questions! Remember that delegating is not just a way to create more...team members by giving them new opportunities. I still need to check myself sometimes to make sure I am...major problem. But I also learned to ask specific questions. “How’s everything going?” is likely to get you...

...Lance-Byrne and Alex Chavez, researchers at the Wyss Institute for Biologically Inspired Engineering. CRISPR/...protospacer. The resulting double strand break (DSB) stimulates DNA repair mechanisms that can be exploited to...are limitations to a system that relies on two distinct Cas9 variants—nuclease-competent or nuclease-null—in...bacterial species, each of which interacts with a distinct gRNA that allows users to decide which Cas9 protein.... When complexed with a truncated gRNA, Cas9 is still able to bind DNA but is no longer competent to cut...

...other split systems where light is required to reconstitute enzymatic activity. Photoactivatable genome ...(Kawano et al., 2015). Nicknamed paCas9-1 and consisting of the fusion proteins N713-pMag and nMagHigh1...far-red light-activated split-Cas9 (FAST) system constitutively expresses the C-terminal fragment of Cas9 while...Michael Lin, who developed a chimeric protein consisting of Cas9 and a photo-dissociable dimeric green... prevent DNA binding in the dark. Upon light stimulation, the pdDronpa moieties move apart, allowing DNA...LOV2 has a C-terminal helix that unfolds when stimulated by blue light. When fused to the catalytic site...

...inactivation of the vector can still occur even in the case of no previous immunity existing for that adenoviral ...challenges remain (Wold & Toth, 2013).  While the pre-existing toolbox for adenoviral vectors in Addgene’s repository... – and we hope that it inspires further use of existing vectors, as well as research into the development...M. (1999). Ovine Adenovirus Vectors Overcome Preexisting Humoral Immunity against Human Adenoviruses In...

...student at Stanford University. Arabidopsis is a fantastic model organism for many reasons, not the least...Agrobacterium tDNA insertion likely precipitated domestication of sweet potatoes (Kyndt et al., 2015)! It sounds...too old to transform. This transformation could still work, but the efficiency will be low. Tips: Use...plants: If you transform early, your plants will still have plenty of time to produce seeds after you expose... plant’s flowers inside. 50 mL falcon tubes or plastic weigh boats are both great options for this, depending...

... during the early stages of the process. Some questions to start with are: Is your sequence of interest...opposite (reverse) orientation? The answers to questions like these will determine which acceptor vectors...assembly efficiency. This can be done through domestication, which is the modification of a DNA sequence... the synonymous mutation of a codon. You can domesticate the sites yourself using PCR, but gene synthesis...gene synthesis, you can request the sites to be domesticated through the process, and request your sequence...

... plasmid is delivered to your target cells, you still need to select for the cells that contain it. Antibiotic... approach in a mammalian cell experiment should still include an antibiotic resistance gene for growth...express your protein of interest. However, you do still need a species-compatible promoter for expressing... consider the viral vectors themselves to be a distinct gene delivery method. We focused this post on ...between prokaryotes and eukaryotes. Conclusion I still think it’s pretty incredible that my cells “speak...