...Yes Agarose gel Table 1: Features of various qPCR chemistries. Adapted from Wong, 2018   ...should be 60-65°C For qPCR, the important primer variable is the annealing temperature, not the melting ...reference genes have shown to have as much as sixfold variation in expression levels reported (Biassoni, 2014)...negative error on the other will introduce unwanted variation into your data. Try different types of pipettes... within each set of triplicates to understand variation due to technical error.  Plate readouts can be...type or tubes, sealing method, and other small variations in consumables. Track and record these details...Reference Dyes Whatever your pipetting method, some variation will occur. A passive reference dye, such as ROX...

... an optogenetics experiment work? What are the various tools and components necessary to run it? And, ...any optogenetic experiment. There are numerous variants for the different types of opsins available and...constructs available through the vector cores of various universities as well as Addgene. See the Deisseroth...cell-type specificity, the kinetics of the opsin variant, and other particularities that best suit your ...these reviews for a discussion of the different variants [1, 2, 4]. And, of course, the best way to learn...learn about the many different opsin variants and how they can be used is to consult the many primary research...has developed many of these AAV constructs with various promoters and other genetic manipulations to help...

... Germline variation contributes to false negatives in CRISPR-based experiments with varying burden across...Beroukhim labs at the Broad Institute of MIT and Harvard, Dana-Farber Cancer Institute, and Koch Institute...the effects of ancestry and germline sequence variation in cancer cell lines took them down unexpected... screens based on the cells’ germline genetic variations. They then wanted to identify cancer-associated...correct, either. However, when they analyzed the variations in gene expression, they found that for approximately...genome does not represent the diversity of genetic variation across the human population. Rather, it primarily...designed that target regions with high genomic variability.   Moving forward It took a five-team response...

...advancing and sharing science! In association with the Harvard graduate student organization Science in the News...public that provides an accessible introduction to various scientific topics. Over twenty graduate student... with the public. The organization runs a wide variety of programs, including fall and spring lecture ...and Reddit AMAs. Their website also provides a variety of short- and long-form written content about timely...co-director Steph Guerra, a 3rd year graduate student at Harvard, who wanted to provide a conference-style experience...and content level. Sponsorship from Addgene and Harvard Integrated Life Sciences (HILS) made it possible... the biomedical sciences and decided to offer a varied format event to keep the audience engaged. The ...

...CRISPR and optogenetic techniques to develop a variety of photoactivatable CRISPR tools. These tools allow...genome editing process. Read on to learn about the various light-controlled CRISPR tools available to researchers...activate gene transcription. The researchers tested a variety of combinations to optimize both fusion proteins...photoactivatable repressor (dCas9). The activity of all variants was reversible and repeatable. Since these initial...CRISPR–Cas9 activity switching via a novel optogenetic variant of AcrIIA4 (CASANOVA). As shown in the figure below...this article). The group first tested photocaging various lysines in Cas9 to determine which best deactivated...Publication System nickname Photoactivatable moiety Cas9 variatn CRISPR protein application Split Systems / Light-induced...

...Here, Liu et al. engineer a chimeric single chain variable fragment (scFv), grafting the binding region of...improve its stability in cells, and use it in a variety of applications. This set of new anti-FLAG frankebodies... (c) Applications of anti-FLAG-FB-GFP labeling various targets with nuclear (top, 4x-FLAG-mCherry-H2B)... Anti-FLAG and anti-HA frankenbodies have a variety of applications, including live-cell imaging, single-molecule...optimizing enzyme activity using an enhanced Cas12a variant (enAsCas12a), minimizing off-target effects by ... reducing recombination by finding acceptable variations in DR sequences. Figure 2: comparison ... Various Addgenies...

... even distinguish between protein variants when used with a variant-specific antibody. TRIM21, an E3 ubiquitin...inactive cas9 (e.g. dCas9) has been fused to a variety of protein modules to repress or activate gene ...complexes to any genomic locus in any cell type. By varying which sgRNAs are used with FIRE-Cas9, experiments...then induced cultures of these transformants with various concentrations of estradiol and progesterone and...transcription factors to control the level of random variation, or noise, in reporter expression. To do so, one... Various Addgenies...

...that don’t utilize NGG   There are plenty of Cas9 variants which utilize alternative PAMs. These alternative...rounds of engineering, they developed the SpRY Cas9 variant (Walton, et. al, 2020). SpRY uses NRN PAMs and ...efficiency and less reliability. Notably, the SpRY variant didn’t display higher off-target effects than SpCas9...experiments. Between SpRY and several of the other Cas variants described, nearly all genomic landscapes should...targeting with near-PAMless engineered CRISPR-Cas9 variants. Science 368,290-296(2020). DOI:10.1126/science.aba8853...PAM compatibility and editing activities of Cas9 variants. Nucleic Acids Res. 2021 Sep 7;49(15):8785-8795...Expanding Beyond Cas9 xCas9: Engineering a CRISPR Variant with PAM Flexibility History of CRISPR Cas – A ...

...unique tropisms are now available. These include variants with broad CNS or PNS transduction, cell type ...  Other model organisms Tropism and efficacy can vary depending on species. LY6A and LY6C1 do not have...Tabebordbar et al., 2021) method, selecting for variants that were effective in C57BL/6J mice, BALB/cJ ...transduces NeuN+ neurons, with transduction efficiency varying from 10-70% in different regions of the brain (... by transferring the 7-mer peptide region from variable region VIII of AAV9-X1.1 to that of AAV1. AAV1...BALB/cJ and C57BL/6 mice and rats. In mice, no variation in transduction efficiency was seen between different...lower than those seen with AAV9, they were highly variable and were not lower than those of AAV-PHP.eB (Goertsen...

...prepared buffer. This is because standard curves vary depending on a number of factors, including the ...equipment, assay incubation time, etc., which can vary even when the same protocol is being followed.  ... to calculate its experimental value. Though it varies from assay to assay, typically the experimental...trendline for the data. The appropriate trendline varies between assays and may be linear or nonlinear. ... of analysis. Different fields and assays have varying criteria for an acceptable R2 but for many scientific...allows you to calculate the curve’s coefficient of variation, or %CV. The %CV of a sample is the standard deviation...the %CV, the more accurate the curve. Though it varies based on the assay, the ideal %CV is less than ...

... different bacteria, and can be transferred to various experimental hosts (Jain and Srivastava 2013). ...structural elements that are versatile and adaptable. Variation in ori structure allows plasmids to utilize diverse...for replication initiation proteins. These iterons vary in their sequence and number and can alter the efficiency... their structural elements, oris interact with various RNA polymerases and proteins from bacterial hosts...proteins (RepA, RepB) necessary for replication in various hosts (Pagatto and Dillon 2001).  The plasmid has...broad host range plasmid with multiple oris that vary in DnaA-box and iteron structure, host-independent...the Broad Host Range Plasmid RK2: Positioning of Various Motifs is Critical for Initiation of Replication...

... can vary significantly depending on transgene design and delivery. To overcome these variations in human...utility of the system for use with low affinity variants of TEV. The new system has been demonstrated to...the evolution platform to generate TEV protease variants. Catalysis by TEV protease allows a transcription...fluorescence protein-based calcium sensors (jYCaMP1), variants of the calcium indicator jGCaMP7, that are optimized...new calcium sensors include syn-jYCaMP1s (slow variant), the Cre-dependent syn-FLEX-jYCaMP1s, and the ... Various Addgenies...

...reach all regions of the CNS. PHP.B, a neurotropic variant of AAV9, is better than AAV9 at gaining access ...PHP.B viruses. Creating PHP.B, a neurotropic AAV9 variant PHP.B’s specificity for C57BL/6J mice is likely...screened in vivo in C57BL/6J mice strains to identify variants that reached the CNS following IV injection. Compared...efficiency, but at a log lower dose than PHP.B.  A Ly6a variant is linked to PHP.B CNS permissiveness Both the ...permissiveness. The Wilson Lab also looked for the gene variant responsible for the PHP.B permissive phenotype ...how the method used to select novel AAV capsid variants could unintentionally limit the utility of candidate...E., et al. "Cre-dependent selection yields AAV variants for widespread gene transfer to the adult brain...

...types or organisms, promoters must be similarly variable. Bacterial promoters only work in prokaryotic ...species from which they were derived. Similarly, the various eukaryotic cell types (mammalian, yeast, plants...gene.  Constitutive  Widespread expression, but may vary by cell type. Tends to resist promoter down regulation...operators. Good for modulating gene expression through varied inducer concentrations.  Sp6 in vitro transcription...well-suited for modulating gene expression through varied inducer concentrations. trp High levels of gene...promoter. Good for modulating gene expression through varied inducer concentrations. Ptac General expression...regulation. Good for modulating gene expression through varied inducer concentrations. Generally better expression...

...owing to its ease of use and utility in a wide variety of organisms.  While much of current CRISPR research... in plants is also being realized. There are a variety of reasons to consider using genome editing to ...for the use of CRISPR in plants, and outline the various plant genome engineering tools that are available...systems, expression of S. pyogenes Cas9 or Cas9 variants (hereafter referred to as Cas9) and a single stranded... the nucleus, and several codon optimized Cas9 variants have been created in an effort to increase translation... Editing Kit from the Stuttmann lab contains a variety of Agrobacterium-compatible, Cas9 containing vectors...plant gene; you can find many plasmids for a wide variety of CRISPR applications in plants available through...

...recombinase variants to optimize PASSIGE efficiency (Pandey et al., 2024). The best-performing variant yielded...TALEs), with fifteen total variants deposited with Addgene. These variants, including a “miniCRISPR” version... linkers and spacers could enable many future variations using nanobodies or scFvs for other targets or...methyltransferase. The strategy is generalizable to a variety of DNA-targeting proteins, so the team created ...allowing the team to develop self-silencing CHARM variants that stop their own expression after silencing...

...neuronal activity. Researchers have designed a variety of photoswitchable elements into many other types...available to disrupt microtubules in cells, they have various disadvantages and limitations – for example, microtubule-destabilizing... Opto-katanin will surely be useful for a wide variety of experiments. Opto-RhoGEFs manipulate cell morphology...other GTPases. The Chow Lab has also generated a variety of membrane-recruitment optogenetic tools for manipulating...feasible or when the protein of interest supports a variety of processes in different cell types. One great...phospholipase D (PLD). Now they present a set of variants called SuperPLDs with 10, 30, 50, and 100 times... and cytotoxicity better than the 50x and 100x variants. But those super-high efficiency constructs are...

...are numerous GFP variants. Here I use the name ‘GFP’, which refers to mEGFP. This variant was made by introducing...jellyfish GFP (AvGFP) to create enhanced GFP (EGFP), a variant with improved brightness (Tsien, 1998). Another...necessary to generate the strictly monomeric EGFP variant, mEGFP (Zacharias et al., 2002). GFP needs oxygen...Cranfill et al., 2016 and FPbase.org). A number of variants remain fluorescent under acidic conditions, with...can be used to detect specific phosphoinositides (Varnai and Balla, 2008) (Figure 2). RNA production can...although it still is worthwhile to generate several variants and check the functionality of the fusion (Mastop...

...London), Dr. Connie Cepko (Harvard Medical School), and Dr. George Church (Harvard Medical School) who took...We've interviewed Addgene depositors who are at various stages in their careers to get advice on choosing..., you're doomed.” 2. Always be sure to include various positive and negative controls to “convince [any...experiment is definitely not a fluke.” 3. Engage in various projects and help colleagues with their work. “...critical thinking skills that are applicable to a variety of careers outside of academia. She has had previous...

...circular plasmid that can be easily maintained in a variety of biological systems. Read on for an in-depth ...backbone. Ideally, the backbone will contain a variety of restriction enzyme cut sites (restriction sites...completion. The time required for complete digestion varies for different enzymes. Many companies now sell ...be cut out of the gel individually. There are a variety of ways to visualize the DNA in your gel (this ...approximately 1:3. Since the number of base pairs for each varies, it is difficult to calculate this based on DNA...for each plasmid you are trying to create, with varying ratios of recipient plasmid to insert. It is critical... plating on the appropriate antibiotic and try varying the “recipient plasmid : insert ratio” in the ligation...