...through the addition of Cre recombinase (Figure 2). Figure 2: A knockout targeting vector designed...difficult to target specifically with a gRNA). Be sure to keep these techniques in mind when choosing a... to knock out exon 2 of a given gene.   Figure 1: A knockout targeting vector designed to insert...for targeting constructs. In the example shown in Figure 1, large sequences corresponding to exons 1 and...-eGFP-PGK-Puro from the Jaenisch lab.   Figure 3: A knockout/knock-in targeting vector designed... and the targeted exon in the gene of interest (Figure 4). When recombination occurs, the gene still functions...cassette has been placed into an intron.    Figure 4: A targeting vector to create a floxed allele...

...long dsRNA via an RNase III enzyme named Dicer (Figure 1). The plant kingdom has expanded its repertoire...involved in the biogenesis of different small RNAs (Figure 1). These siRNAs can exert their gene silencing... modifications of complementary gene sequences (Figure 1). As a result, gene silencing can be easily induced...from the ease of use in dsRNA applications. Figure 1: Schematic of an RNAi-inducing transgene. Typically...that facilitates the formation of a hairpin RNA structure, resulting in processing by the endogenous RNAi...greatest advantage of RNAi is the straightforward nature of the technique—all that is required dsRNA complementary...complementary to your gene target as inverted repeats (Figure 1). With this technique, multiple genes can be ...

...halt degradative processes while maintaining the structure of your samples, the relationships between cellular...that occurs depends on the incubation time and temperature, so you may be able to recover good antibody ...cross-linking, but it doesn’t preserve sample structure quite as well as a cross-linker. This is because...fixation with these chemicals, which impacts sample structure and could also cause your target to be washed ...Furthermore, these chemicals can also alter the structure of proteins, which could disrupt your target epitope...good thing depending on your experiment.   Figure 1: Choice of fixation method impacts antibody binding...dehydrates the protein, which can disrupt protein structure and thus structural epitopes, but can reveal linear...

...vectors effectively. Retroviral structure   Figure 2: Structure of a retrovirus. Image re-used ... you can use Figure 1 to compare the genomes of these two family members.   Figure 1: Gamma-retrovirus...2021 under CC-BY license. Figure 2 gives a detailed view of the structure of a retroviral particle, which...   Figure 3: The life cycle of a retrovirus. Image courtesy of Jeremy Luban, MD. Figure 3 depicts...vectors correctly. A solid understanding of the structure of a wild-type viral particle — and of the normal...depicts the viral life cycle. One should ponder this figure often, especially the three steps from the 12 o...

...bacterial recombination pathway to allow any cloning procedure to be performed using a simple two-step, 2 hr ...hr protocol prior to transformation (Figure 1). As this approach requires no special reagents or purification...be immediately adopted by any laboratory. All procedures are performed using the same three step protocol...cells. Primer design Using specific primer design (Figure 2), IVA cloning can be used to perform any plasmid...design for each type of modification is shown in Figure 2, and is discussed in more detail below. The main... the template binding region, with a melting temperature (Tm) of around 60 °C for all primers. The length...assembly. Combining multiple modifications A key feature of IVA cloning is the ability to combine multiple...

...fluorophore at the respective subcellular structure (Figure 1). Here, MXS Chaining gave researchers the...various mammalian promoters), the same cloning procedure can be used to incorporate each element into the...These fluorescent reporters can be used to detect structure and protein localization, as well as cellular ...with MXS-chaining were used to label subcellular structures in live cells. Four individual expression constructs...fluorophore, and thus enabled labeling of particular structures (Table 1). Each construct was flanked with a ...final construct was then introduced to HeLa cells (Figure 1). The resulting HeLa cells show robust labeling...Table 1: MXS Constructs for Labeling Cellular Structures Construct Fluorophore Excitation/emission...

...fusion proteins can visualize: Cell Structure Plasmids: Cell Structure Plasmids: ER mCherry-Sec61 ...domain. Feng Zhang's group utilized the bi-lobed architecture of Cas9 to engineer a series of "split" cas9...for iLID nano and 800nM to 47µM for iLID micro; Figure 1). With this great affinity range, these LIDs ...light-mediated subcellular localization in mammalian cell culture and reversible control of signalling pathways. ... off and on your favourite signalling pathways (Figure 2). They are all available now at Addgene. 1...efficiency and gene expression. The pUCXKT vector ensures that all screened plasmids contain a gene variant....g. the ER) and a reporter protein of interest (Figure A). Franck Perez’s lab has engineered hook proteins...

...toolbox. We'll cover how Cas13a was identified, the structure and function of Cas13a with a focus on what makes...genes that fell into 3 categories based on the architecture of the CRISPR protein in question: C2c1, C2c2... referred to as Cas13a) is unique in terms of structure and function, and will therefore be the focus ...cleaves RNA rather than DNA substrates. In terms of structure, Cas13a shares no homology to the most commonly...CRISPR Blog Posts Applications of Cas13a   Figure 1: Using Cas13a as a diagnostic tool. A pool of...applications of Cas13a given what we know about its structure and function? For starters, Cas13a can be used...2017). This system, dubbed SHERLOCK (depicted in figure 1) has been used to differentiate strains of Zika...

...Zhang, a Ph.D. candidate in the Yang Lab, Qi Li, a lecturer at Sichuan Normal University, Jieze Zhang, an ...plasmid that expresses the S. pyogenes Cas9, a temperature-sensitive replicon for self-curing, an arabinose... now take place. After editing, pTargetF can be cured from the cell by inducing expression of the sgRNA...other edits. Once you have all the edits you want, cure pCas9 by growing the cells at 37 °C.     The new...us to update the pCas/pTargetF system.   Figure 1: The editing process begins with the addition...DNA for editing to take place. Then plasmids are cured from the cell. Steps 1-1 and 1-2 can be done simultaneously..., 2) changed the replicon of pCas into a non-temperature-sensitive replicon, and 3) added the sacB gene...

...required longer exposure time than its protein equivalent and was less stable due its RNA nature, it paved ...including: Environments with higher temperatures, since proteins can denature while aptamers are more stable...form the base of the stem in an RNA stem-loop structure (see Fig. 1a). When the aptamer binds its ligand...laboratory took advantage of their deep knowledge of the nature of GFP fluorescence to create an improved fluorescent...fluorescent aptamer system. Knowing that GFP’s  structure stabilizes a 4-hydroxybenzylidene imidazolinone...cells. The new aptamer also has a higher melting temperature than its Spinach ancestors, and has a lower magnesium... its biosensor capabilities (see Fig. 1b). The future of aptamer fluorophores The Jaffrey lab and others...

.... These features enable the fibers to emit light for optogenetic purposes, measure temperature, study ... described, then measured electrophysiological potential afterwards. This measurement trended with expression... the fibers would need to be, as well as other features necessary for implantation in brain tissue. The...the wild west for fiber implants. Due to the soft nature of the intestines and the requirement for digestive...multiple channels and can also record tissue temperature proximal to the implanted fibers. As important...Alternatively, the system could be used to deliver or measure new variables. The sky is really the limit for ...wireless modulation of gut and brain neural circuits. Nature Biotechnology, 1–13. https://doi.org/10.1038/s41587...

...amplification and verification processes we use to ensure high-quality library distribution. You can also...library contains many plasmids, care must be taken to ensure that all of these plasmids are amplified and none...First, use enough DNA and competent bacteria to ensure all components of the library are represented. ...performing amplifications, and we use electroporation to ensure a good transformation efficiency. For a typical...Addgene still recommends plating the transformation mixture on solid media. The colonies are then scraped off...we created for the Brunello library is shown in Figure 2. You can use a small PCR product because the ...AUC of 0.5 representing perfect uniformity (see Figure 3A). Addgene finds that this representation is ...

...experimental settings: HEK293 cell culture and in live zebrafish. For cell culture experiments, only dL5**-expressing...just 1 minute of exposure. MG-2I-dL5** experienced minor bleaching after longer exposure times. Additionally... fluorogen that can be used to ablate cells in culture and in vivo.  Read on to learn more about this ...exposed to light stained dead within 30 minutes of exposure. 90 seconds of illumination were sufficient for...mCer3 cells present 96 hours post treatment (see figure 2). Induced cell death was confirmed by TUNEL staining...light. They found that KillerRed required longer exposure times (5 minutes vs 10 seconds) to achieve the...genetically targetable near-infrared photosensitizer. Nature Methods,13(3), 263-268. doi:10.1038/nmeth.3735....

...(rAAV) vector (Figure 1). Everything else outside the ITRs gets left behind.    Figure 1: Recombinant...polyA signal. Created with biorender.com. ITR structure  ITRs are symmetrical 145-nucleotide sequences...palindromic arms (A-A’, B-B’, and C-C’) that give the feature its characteristic T-shape. The arrangement of ... the ITR, which can either be “flip” or “flop” (Figure 2). Additionally, the ITR contains a 4-nucleotide...target site for Rep proteins (Savvy et al. 2013). Figure 2: AAV2 wild type ITR in flip and flop orientation... B-B’, and C-C’ regions) and forms a T-shaped structure. The Rep binding element (RBE) and terminal resolution...FAQs Due to their high GC content and hairpin structures, ITRs are prone to replication errors and disruptive...

...prior to your departure for the new placement. Make sure you read any relevant literature for the techniques... have structured visiting positions within defined labs which you can apply to. These structured types...institute and mentor that they may want to consider for future work placements. The chance to learn and collaborate...prior to arrival so that you can work together to ensure your expectations are reasonable, your reagents...Unless some of these logistics are covered by a structured visiting program, you will have to make arrangements...get the ball rolling! If you are applying to a structured program, they likely have guidelines to consult...don’t make your bench mentor wait on you!), make sure you are prepared (read any protocols the day before...

...many available to choose from) is now a common feature in reporter plasmids.  Learn How Luciferase Can... reporter protein or its enzymatic activity is measured. The effect of promoters or enhancer regions on...case of a luciferase assay, photon emission is measured resulting from the catalysis of a chemical reaction... Green Fluorescent Protein (GFP) because of the nature of the chemical reaction compared to using a high-intensity...improved signal sensitivity since photons are simply measured – they are not required to initiate the reaction...Expression of luciferase as the reporter gene is measured via its activity to produce light, often with ...luminometer or scintillation counter, and the activity measurement can then be used to quantify the effect of the...

... from viruses that infect people in nature, extra safety measures, like replication incompetent viral ...the stomach flu), and many more. Their infectious nature allows them to easily deliver their “cargo” to ... make you sick…well, you feel it. But the same features that make these familiar viruses such a pain is...biological tools.   Viruses as biological tools In nature, viruses infect cells and organisms (which we refer...of cell the virus will target and fuse with. In nature, this determines which organisms and cells they...Institute. Its genome, however, isn’t enough to ensure the virus can replicate and spread. Instead, viruses...are required. We will be covering these safety measures in a separate, upcoming post. If a viral vector...

...yeast, are temperature sensitive screens. These involve raising or lowering the culture temperature to exacerbate...Selections do require there to be a culture condition that can apply enough pressure to select for variants of ...individually administered and then outcomes are measured. While the entire genome can be knocked out with... have a screen that doesn’t yield any hits. Make sure you plan ahead and consider these questions to save...though. A selection can take the form of varied temperature, nutrient availability, drugs, and much more....with screens. Selections require there to be a culture condition which eliminates manipulations that do...conditions which convey resistance to a drug. You could culture cells in the drug of interest at a lethal dose ...

...perturbation The Penn State Protein Ladder Engineered miniature CRISPR-Cas system for mammalian genome regulation...frankenbodies include mEGFP, mCherry, and HaloTag. Figure 1: (a) Anti-FLAG frankenbody (FB) construction ...advantageous to Cas9’s larger expression requirements (Figure 1), but Cas12a has generally been underutilized...finding acceptable variations in DR sequences. Figure 2: comparison of Cas 9 vs. Cas 12a expression cassettes...optimized toolkit furthers Cas12a’s potential in future genetic screens.   DeWeirdt PC, et al. Nat Biotechnol...commercial ladders which also provides a useful yet low-pressure teaching opportunity for lab members or students... to protein expression and purification. Figure 3: The Penn State Ladder run on SDS-Page gels. ...

... conjugated antibody. Compensation beads are a mixture between synthetic beads that can bind to a conjugated... your voltage using your single color controls (Figure 1A), instead of using your actual samples. They...the settings with experimental samples. Figure 1: Example flow plots for a single color control... may otherwise be difficult to separate out. In Figure 2, you can see that, without the FMO, it would ...population from the Ly6C positive population. Figure 2: An FMO gating example. The FMO sample (A) can...clear positive and negative population. If you’re unsure what your population will look like for a particular...provided by the antibody companies or check the literature. Pro tip! Early gates, like your live/dead markers...