...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...

... 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...

...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 ...

...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...

...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 ...

...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...

...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...

...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...

...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...

...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...

...How are inducible promoters regulated? Inducible promoters can be regulated by positive or negative control...Addgene! Chemically inducible promoters Chemically regulated promoters are among the most common inducible ...protein purification. When arabinose is absent, regulatory protein AraC binds O and I1 sites upstream of...complexed with cAMP activator protein (CAP) can also stimulate AraC binding to I1 and I2 sites. Supplementing...include positive inducible alcohol and steroid regulated promoters commonly used in plant research. ...Example Activator Addgene Plasmid/Kit Alcohol-regulated AlcA promoter AlcR pGGA008 (AlcA) and pGGC011...AlcR) from red flame GreenGate kit Steroid-regulated LexA promoter XVE (synthetic) pFZ19 (yellow flame...

...externally. Fruit flies are also relatively straightforward to manipulate genetically. Scientists can make...make it easy to study the effects of genetic manipulations. In this blog post, we’ll cover five popular... model organism for scientists as they have a relatively short generation time for mammals - the time ...study diseases such as cancer since mice better recapitulate the complex interactions between cancer cells...are genetically tractable. Mice can be easily manipulated with tools like CRISPR to make transgenic lines...entirely sequenced and is very amenable to genetic manipulation. Yeast cells are great model organism not only...studied in other model organisms that better recapitulate in vivo conditions similar to humans. For example...

...antibodies bound to an ELISA plate to capture Myc-tagged sybodies. Then, the biotinylated target protein is added...coronaviruses into a llama over several days and isolated special camelid antibodies that bind to the spike...secondary nanobodies blog post.  A rapid selection platform for generating sybodies against membrane proteins...weeks (Zimmermann et al., 2020). This selection platform was designed so that any standard lab can quickly...in the form of mRNA for academic research. The template plasmids for generating your own libraries are...Generation Toolbox! Starting with libraries and a biotinylated (for purification purposes) protein target of...involves using a commercial kit for in-vitro translation of the mRNA libraries. Each mRNA molecule in ...

...gene drives” capable of spreading through wild populations. Whereas most genomic changes impose a fitness...could eventually spread through the entire wild population with unpredictable ecological effects. Because...chromosome and its own gene is used as a repair template, thereby converting the heterozygote into a homozygote...building gene drives that can spread through populations of transgenic laboratory organisms but not wild...sgRNA-only drive will spread exclusively through populations that already express Cas9 from an unlinked locus...sequence will only spread in transgenic laboratory populations with that sequence2. Both methods are easy to...performed in regions that don't have resident populations of the relevant mosquito species. Reproductive...

...expression, and protein translation. Its genome consists of “early” (E) and “late” genes, and the transition...transition between early and late infection (and therefore between early and late gene transcription) is demarcated...unusual cases of acute hepatitis in the pediatric population. Once ~400 cases had amassed in places around...exist. The life cycle of AdVs is dependent on modulation of the host cell’s cell cycle; inhibition of ...recombinant sequence (transgene) instead.    Although the late genes are usually present in the transfer plasmid...expressed, since the early genes are absent, and the late genes (and their structural proteins, which would... some of the wild-type viruses can persist as a latent infection in certain tissue reservoirs (Pereira...

...optogenetic stimulations, they simultaneously recorded the electrical impulses of discrete subpopulations of this... or reward-related behaviors. The high spatio-temporal precision and reversible modulation of FLEx vectors...combined optical and genetic methods to photo-stimulate mammalian neurons and alter synaptic events: a..., 2005; Deisseroth et al., 2006). Despite its relatively recent development, the history of optogenetics...rhodopsins and neurons have in common? Neuronal stimulations are triggered by the movement of ions across...Zhang, 2012; Towne et al., 2016). Optogenetic neuromodulation is made possible by the light: when hit with...viral-infected neurons are subsequently photo-stimulated through fiberoptic cannulas directly implanted...