...expression. Transgenic and knockin mice As the name suggests, transgenic and knockin mice contain an additional...Conditional knockouts or knockins A subset of transgenic mice, conditional knockout/knockin mice, use the Cre-lox...construct incorporated randomly into the genome, knockin mice have the gene construct inserted at a specific...DNA randomly into the mouse genome. In contrast, knockin strains are generated via homologous recombination...breaks, such as ZFN and TALEN, can used to engineer knockin mice. Co-injection of a targeting vector containing...based systems have been developed for generating knockin mice but because CRISPR/Cas9 engineering favors...following Cre-mediated recombination. In conditional knockin mice, the target gene is initially inhibited by...

...cutters. You can also sort enzymes by clicking on column heads. Clicking on a particular restriction enzyme...GSL Biotech's SnapGene Server Software. With the backing of SnapGene’s sequence viewer software and extensive...Maps This is the first tab you’ll land on after clicking the “Analyze Sequence” button. The maps hosted...similar hover over features. As with the map tab, clicking on two separate locations in the linear sequence...Types listed are standard GenBank feature types. Clicking on a particular feature here also highlights that...binding site locations, lengths, and direction. Clicking on a particular primer here also highlights it...updates in the comments section below and keep checking in at blog.addgene.org for additional enhancements...

...tool of choice. Nicking sgRNAs PE3 and PE5: These tools use an sgRNA to guide nicking of the unedited...editors bound to sgRNAs only drive nicking (right). Start with nicking sgRNAs ~50 bp from the prime editing... Figure 7: Nicking sgRNAs can be designed to target edited DNA, so that nicking only occurs after... prevents the Cas9 nickase from re-binding and nicking the newly synthesized strand before heteroduplex...BioRender.com. PE3b and PE5b: In this approach, the nicking sgRNA is designed to bind only after the edit is...

...making the detection of rare events difficult. Dickinson et al. sought to create a new and efficient selection-based...Figure 1: Self-excising cassette schematic from Dickinson et al. (2015). Used with permission from the Genetics...for the targeted gene. After initial testing, Dickinson et al. tested this system across 7 genes in parallel...modification. Figure 2: Schematic from Dickinson et al. (2015) illustrating the steps to fluorescently...Society of America. Applications of this system Dickinson et al. have developed a modular system that requires...with a Self-Excising Drug Selection Cassette. Dickinson DJ, Pani AM, Heppert JK, Higgins CD, Goldstein...using Cas9-triggered homologous recombination. Dickinson DJ, Ward JD, Reiner DJ, Goldstein B. Nat Methods...

...scanned to verify that it is the correct sample. Tracking samples - it's all about location, location, location...it could take us hours just to find one sample! Luckily, if a rack of tubes is dropped at any point in ...in house if necessary.   Tips for barcoding and tracking samples 1. Start with the tube - Barcoded tubes... information in a small space and is ideal for tracking large collections. Many companies, such as Thermo...Although we’ve developed our own computer system for tracking samples, it can be done easily with a barcode ...multiple people or even multiple labs. Careful tracking prevents us Addgenies from wasting hours of valuable... sample tracking...

...unused surface-binding sites in the wells with a blocking protein such as BSA, followed by several wash ...

...improve resolution, use a stacking gel on top of the resolving gel. A stacking gel typically has a different...buffer, blocking the membrane  from binding to the antibodies during the stain. The ideal blocking buffer...of the proteins in a sample migrate through the stacking gel at the same pace and enter the resolving gel...include suggestions for working concentrations, blocking buffers, and incubation times. If the vendor includes...

...available for tracking certain cell types. Another useful tool for marking and tracking cells is the the... Pathogens - Kamenyeva et al, 2015. Cellular trafficking studies enabled by fluorescent proteins The optimization...that some are more reliable and more efficient at tracking cells. Good two-photon probes when using a standard...photoactivable or photoconvertible proteins, as it enables tracking throughout the lifetime of the cell.     The past... cellular trafficking...

...monitoring the first round of translation or by tracking the translation of a single mRNA over time. Both...translation. Single molecule continuous translation tracking TRICK allows you to see the first round of translation...translation, but it doesn’t allow continued tracking of a given mRNA because NLS-PCP-GFP is removed by ...first ribosome that translates the mRNA. To permit tracking over time, Yan et al. devised a system that labels...keeps it in a single field of view for constant tracking. You can then directly monitor multiple rounds...

... account, you can begin the deposit process by clicking "Submit Online Deposit" (for online submission... menu: Figure 1: Begin the deposit process by clicking "Submit Online Deposit" or "Download Deposit Spreadsheet...  Figure 4: Add plasmids to the article by clicking “Add Plasmid to this Article” (left) and entering...button and jump around to the different pages by clicking on the appropriate boxes at the top of the page...the plasmid to copy from the drop-down menu and clicking the white "Copy" button. *Note: The "Copy" button...Submit Deposit" button to complete the data entry. Clicking the "Submit Deposit" button will allow you to ...

... have fused with the vacuole or a lysosome are trickier to identify because their contents can be at various...autophagosomal membranes, so tagging it with GFP allows for tracking the localization or accumulation of pre-autophagosomal...an autophagy-independent manner. Additionally, tracking Atg8p or LC3 doesn’t provide information about...easier to study the mechanisms behind autophagy by tracking what’s being transported to the yeast vacuole ...vacuole (i.e. cytosol, mitochondria), rather than tracking generic autophagy markers. It’s particularly useful...

... CRISPR-based approaches for the generation of knockins via homologous recombination with a homologous...application - the creation of sequence-specific knockins - for which the AAV approach is most well suited...suited, since it routinely generates knockin efficiencies of 1-40% with G418-selected clones. However, there... CRISPR-based approaches for the generation of knockins, it is not always ideal. Because of these limitations...essentially all of an AAV’s ~4.5 kB packaging capacity. Luckily, researchers have devised methods to deliver CRISPR...

...fluorescent proteins to oligomerize is known as “stickiness.” Since the fluorescent tag should operate as... per unit.  There are several ways to measure stickiness. In vitro, ultracentrifugation or gel filtration...oligomerization in cells. In addition to measuring stickiness, several researchers have also collected data...characteristics are important, but have less priority than stickiness. In my experience, fluorescent proteins that ...independent of GDI-mediated extraction and actin-based trafficking." PLoS biology 13.4 (2015): e1002097. PubMed ...

...authors’ compared nicking the top strand first followed by the bottom strand versus nicking the bottom strand...with a twist since it relies on the sequential nicking, degradation, and PCR-generation of a mutant version...Supplementary Note 1 in Wrenbeck et al for details on how nicking was optimized. How should I design my mutagenic...gene of interest has a BbvCI site(s). If so, the nicking mutagenesis protocol will still work if the orientation...

.... You can find Addgene's sequencing results by clicking on the "View Sequences" link on the Plasmid Information...

...edit and homology to the genomic DNA locus. After nicking by Cas9, the homologous pegRNA sequence hybridizes...previously used when they developed base editing. By nicking the non-edited strand, they can cause the cell ...efficiencies 2-3-fold. However, the process of double nicking increases indel formation slightly. Designing the...the edited strand, as in the PE3b system, guides nicking of the unedited strand only after editing has occurred... ✓ ✓ ✓   ✓ Second sgRNA to guide nicking of the unedited strand Favor the edited strand...

...biological motion (whether endocytosis, vesicle trafficking, or muscle contractions) are produced by orchestrated...were first expressed in E. coli in the 1980s  (Kanabrocki et al 1986). Much later, it was shown that putting...her on Twitter @jessicapolka.     References 1. Kanabrocki, J. A., Quackenbush, R. L. & Pond, F. R. Organization...

... microcentrifuge or falcon tube. GENTLY mix by flicking the bottom of the tube with your finger a few ...

...ligase, 5min at room temperature is enough. For trickier ligations (such as ligation of annealed oligos...

...ever-growing variety of recombinase systems. Picking Ligands Picking the right ligand is also important for ...blood-brain barrier.  Another consideration when picking ligands for chemogenetic studies is whether or ... are viable for translation to human patients. Picking receptors The receptor you choose is largely dependent...