... Single-stranded DNA repair templates Short single-stranded DNA templates have been shown to improve the...laboratory mouse. It’s also made targeted insertions relatively simple in C. elegans and bacteria. But CRISPRing...from low efficiency when a standard dsDNA repair template is used. The original design for creating a conditional...targeting two intronic regions and two repair templates in order to insert two loxP sites. Since two double-strand...ameliorate this problem. Briefly, you design your donor template to include a T7 promoter upstream of the left ...vitro transcription, and use the IVT product as a template for reverse transcription to create your ssDNA...promoter will not be included in the final ssDNA template. Although effective, ivTRT may be more challenging...

...example Tuller et al. found that translation efficiency is well correlated with codon bias in both E. coli...therefore may need fewer resources for the entire translation process. For example, during high growth rate...rate conditions, E. coli preferentially upregulates production of tRNAs that recognize codons found in highly...tRNAs, different regions of the protein may be translated at different rates. The ribosome will move quickly...the ribosome stalls, this may give the swiftly translated regions a chance to fold properly. For example...with specific secondary structures in 10 closely related yeast strains. Adaptation to changing conditions...codon preferences online). Even if the gene is translated, it may not function properly. This is the result...

... constructs. In multiple HIV-1 latent T cell lines, as well as a latent microglial line, treatment with...doesn’t completely disappear; it just hides in latently infected cells. To truly cure HIV-1, researchers... levels due to the viral reservoirs located in latently infected cells. Although ART-treated patients ...disease. This increased disease risk is attributed to latent HIV reservoirs, chronic inflammation, and negative...doesn’t see any threat to which it should respond. Latently infected cells evade immune detection because ...first, “shock and kill”, aims to reactivate the latent HIV so that infected cells will produce viral proteins...joined together. Clonal Cas9/gRNA-expressing cell populations were also immune to HIV-1 reinfection. Importantly...

... are enriched relative to controls using Split-BioID-GFP and BioID run on six unrelated proteins. One ...These interactions are often modeled as binary relationships, i.e. protein A interacts with protein B; but...controllable version of the proximity-dependent biotinylation technique BioID. The key advantage of Split-...BioID, BirA* is fused to a bait protein and it biotinylates proteins that are within a ~10 nm radius of ...biotin. When proteins Y and Z part ways, BirA* biotinylation activity ceases. After biotin tagging of proteins... PPIs are identified via mass spec analysis. Biotinylated proteins are considered possible interaction...different levels of fusion protein expression and biotinylation depending on which terminus of the bait proteins...

...promoter or the coding sequence and the template or non-template strand, among other parameters. .  Stanley...introduced along with an oligonucleotide repair template. Through recombineering, the locus of interest...interest is modified to match the repair template, and the locus cannot be recognized by the spacer-derived ... contains the specific gRNA(s), and the repair template is supplied as a dsDNA fragment. Each round of...with XbaI to insert extra elements, like a repair template, using Gibson Assembly or restriction enzyme cloning...does not function in bacteria, most efforts to regulate gene expression were limited to inducible promoters...CRISPR offers a much more user-friendly way to modulate gene expression. The Marraffini lab and Qi lab...

...relying on correlations between actual antibody labeling and expected labeling. Correlation cannot prove...practice to combine correlation with other approaches when possible. Finally, correlation of -omics data ...a powerful tool to delineate cell populations and to study relative protein expression. Its reliability...against markers of the human blood leukocyte populations, as well as a pragmatic approach to determining...and the abundance of the epitope in the sample, relative to cross-reactive antigens – that is, an antibody...images showing the staining intensity in the KO population (pink histogram) compared to the WT cells (green...shows separation between the wild-type and the KO population (Figure 1), further optimization can be performed...

...the template. The opposite is true if the sense strand is nicked for template formation: template will...low primer-to-template ratio is used to promote annealing of one primer to each template. The PCR product...Error-prone PCR: This method amplifies the wild-type template with a “sloppy” version of PCR that results in...). It uses a uracil labeled wild-type ssDNA as template for a site-directed mutagenesis PCR that uses ...strand (step 2 and 3). Then the uracil-labeled template strand is degraded by uracil DNA glycosylase (...present on both strands of the plasmid. The ssDNA template is generated by transforming a backbone phagemid...days to generate the phage-derived uracil labeled template, this method requires further DNA shuffling to...

...the blocking-only templates will compete with the blocking/intended mutation templates resulting in cells...simultaneously introducing a homologous DNA repair template, such as a sin­gle-stranded oligodeoxynucleotide...In mammalian cells such as stem cells, HDR is relatively rare and DSBs are predominantly repaired by NHEJ... very excited to set up a CRISPR-based editing platform in our lab. Our goal was to use the system to ... – Edit the cells a second time using a repair template that corrects the blocking mutation and again ...that the cell was only using a part of the repair template and mutations further away from the cut site were...characterize this phenomenon and found a general relationship between the distance of a mutation from the ...

...multicistronic vectors, and post-translational regulatory elements. Examples of these approaches are given...regions, neuronal networks, and specific neuronal populations to consider. To tease apart how different neurons...can combine classical genetic tools with the manipulation of specific neuronal subtypes and determine ...combinations to choose from for your experiments! Late last year, we hosted our first AAV Education and...-based molecular tools for targeted neuronal manipulation. The goal of the workshop was to share the results...for neuroscience see Table 1 from the paper. 2. Manipulate your cell of interest: Cell-type specific expression...achieved in nontransgenic animals by using cis regulatory elements such as promoters and enhancers, multicistronic...

... our high-throughput DNA isolation process that yields high-quality isolated DNA samples of sufficient...process is completed in a plate format yielding anywhere from two to six plates of 96 samples per week. ...anyone who has ever worked to sequence DNA on NGS platforms will tell you, while the process has become much... process only takes about one day, even for six plates (576 individual plasmids)! These libraries are ...perform Picogreen quantification on each and every isolated plasmid sample and normalize each to fall within...of this range are “failed” and removed from the plate to be prepped again. These holes are then filled...mutations, we check to see if they affect the translated amino acid. We also confirm that the species ...

... precision for relative quantification. A consistent 5% positive error across the plate is fine; a 5% ...up your plate in a way that will tell you if you have pipetting errors on the edges of the plate. Compare...variation due to technical error.  Plate readouts can be affected by plate type or tubes, sealing method, ...of mouse prominin, is blood glucose-regulated and can up-regulate the expression of GAPDH. Biochem Biophys...You know qPCR is most commonly used to measure relative quantification of mRNA, which can be used as a...reported to work well in the Lightcycler while being relatively easy to multiplex (Thellwell, 2000). See Table...possible to multiplex your reference genes to save on plate space. For many cell lines and tissue types, appropriate...

...transformation plate can be manipulated to result in appropriate levels of colony plating densities. Ligase...Outgrowth plated) Correct assemblies per plate Fidelity of assembly (percent correct) Calculated colony ... can plate larger volumes of the outgrowth containing the transformants onto the selection plate. Figure...of assemblies per plate using outgrowth volumes described in Figure 3, with calculated yields from entire...phenotype upon growth on LB/Cam/X-gal/IPTG agar plates, indicating successful reconstruction of the coding...assemblies was achieved by the sequencing of plasmids isolated from blue or white colonies. Sequencing of blue...Figure 2 shows representative transformation plates obtained from 1-, 12- and 24-fragment assemblies of ...

... the cumulative number of labs who have deposited CRISPR plasmids (Figure 1C), and the cumulative number...distribution and deposits. (A) Cumulative CRISPR plasmids shipped by year. (B) Cumulative CRISPR plasmids deposited...(C) Cumulative number of laboratories that have deposited CRISPR plasmids by year. (D) Cumulative CRISPR... a classic double-stranded break, while modulators regulate transcription and chimeric efforts make a ...Distributing and depositing In Figure 1, you can see cumulative numbers of CRISPR plasmid distribution and deposits...nuclease into three functional groups: nucleases, modulators, and chimeric effectors. Nucleases generate a...distribution and deposits per year, rather than a cumulative total.    Figure 2: Addgene CRISPR deposit...

...used to calculate the concentration of the sample. This concentration can then be used to calculate the viral...generator to the PCR plate. Maintaining a high droplet count is important for calculating the concentration...Addgene, we typically load 3 dilutions onto our ddPCR plate. As the titer is unknown, each dilution should fall...making the dilutions, they are transferred to a new plate containing the mastermix which includes primers ...a droplet reader extracts the droplets from the plate and measures the fluorescence amplitude of each ...positive (blue) and negative (gray) droplets. The no template control wells should have very few positive droplets...approximately 1 positive copy per microliter in the no template control wells. You’ll also notice a reduction ...

...are sigmoidal. Once the trendline is calculated, you can extrapolate the concentration of the unknown sample...software to calculate the data’s coefficient of determination, R2. R2 is the square of the correlation between...could be because there is no correlation or it could mean that the relationship is non-linear and you should...versus the response and use that information to extrapolate the concentration of an unknown sample. For example...the accuracy of the curve, which we will discuss later.       Standard curves must be included every time... the experiment and use the standard curve to calculate its experimental value. Though it varies from ...measurements and the trendline y = 0.6561x + 0.019 calculated. The concentration of an unknown sample is determined...

...detected on commercial lateral flow strips, similar to pregnancy tests. With lateral flow strips, the presence...recently adapted the DETECTR platform to detect SARS-CoV-2 using a lateral flow strip format. They published... to develop simple, portable, and inexpensive platforms to reliably detect nucleic acids at the atomolar... to cleave ssRNA or mRNA. It also exhibits a “collateral effect” of non-specific ssRNA cleavage. How does... for nucleic acid detection almost anywhere as lateral flow strips are easy to transport and work rapidly...reliably distinguish between Zika and a closely-related virus, Dengue from multiple sample sources. SHERLOCK...from cell-free DNA fragments as well as health-related single nucleotide polymorphisms (SNPs) from human...

...we shared materials and equipment between labs. Later I worked with people outside the department to accomplish... connect the imaging community and to create a platform for discussions and potential collaborations. ...loads of opportunities to create communities, platforms, and to be a part of projects that connect people...realize. Being at a university provides the perfect platform for practicing initiative and for training in ...conference calls and make sure to maintain these relationships by keeping in touch by email and social media...media (see the section on “Maintaining Relationships” below). Expand: Collaboration opportunities outside... fun. I just had to remember to approach the relationship with the perspective of my potential collaborators...

...antitoxin; Modulation of killing (mok), required for hok translation. Here, Mok and Hok translation is coupled...that is regulated by antisense RNA, which base pairs to cognate mRNA to inhibit its translation. The hok...coupled. Translation from the Shine-Dalgarno (SD) sequence of mok results in translation of Mok and Hok...restriction-modification systems onto plasmids allowed cell populations to retain more of these plasmids as compared ...the cell grows resulting in the appearance of unmethylated sites in the chromosome. The more stable endonuclease...chromosome leading to cell death. Antisense RNA-regulated systems Toxin-antitoxin systems are not just restricted...hok/sok system is the first antisense RNA-regulated toxin-antitoxin system discovered. Unlike the systems...

...antigen present in the sample and is calculated by extrapolating from the standard curve. Because the ...antigen present in the sample and is calculated by extrapolating from the standard curve. Scientists often...same overall process. First users coat a multiwell plate with a capture reagent. The capture reagent may ...of the sample to that of a standard curve to extrapolate the concentration of antigen in the sample. A...substrates since they can be read on a standard plate reader as opposed to chemifluorescent, and chemiluminescent...antibody binds to antigen coated on the multiwell plate. In an indirect ELISA a primary antibody first binds...primary antibody. In a sandwich ELISA the multiwell plate is first coated with a capture antibody that binds...

...undergo significant post-translational modifications including glycosylation, disulfide bond formation...proteins functionally relative to the untagged protein of interest. To avoid the accumulation of dark fluorescent...is, unfortunately, some investigators realize too late that they have missed the impact of dark, non-fluorescent... fold? Misfolded FPs do not fluoresce. A dark population is not readily apparent and can confound quantitative...construct [1,2], i.e. positioning of the FP sequence relative to cell compartment targeting sequences, which...1) [4,6–8]. Additionally, FPs that encode N-glycosylation consensus sequences are modified with the addition...addition of N-glycans [9]. Both of these posttranslational modifications can disrupt FP folding rendering...