...researchers new to GCE aren’t able to successfully complete their experiments and many others are dubious ...’t give it a try. Optimization Why is GCE so complicated? Well, while the techniques are straightforward...tools are not very robust. When using GCE, each component of the experiment system (including media, cell...optimization parameters the same for all three components, meaning that the final protocol may involve ...has “10x the impact of PCR. But it’s 100x more complicated.” And Ryan Mehl would know. He’s the director...

...Cas enzymes and variants available. Without a comprehensive resource, it can be overwhelming to choose a...classification system, CasID, to help facilitate direct comparison of different enzymes. CasPEDIA is meant to be...how can I multiplex this enzyme? What other RNA components are required? CasPEDIA provides information on...gRNA design, etc.). With this feature, you can compare different Cas enzymes to make sure you are getting...isn’t always the winner!).   Fig. 3 – Comparison of different Cas enzymes using CasPEDIA. Adapted...

...be incorporated into these constructs without compromising cleavage efficiency. In addition, tRNAs contain...generation for two PTG constructs in rice protoplasts. Compared to matching traditional gRNA-expressing constructs...examined editing efficiency in intact rice plants, comparing traditional gRNA constructs with PTGs. In PTG ...frequency at both targets (35% and 76% biallelic), compared with only 44% and 66% indel frequency with the...in the graphic below - including some simple comparisons. The easy multiplexing afforded by PTGs will...

...multi-protein complex, stabilized promoters can eliminate variability between the different components. When ... origin of replication will be overrepresented compared to those further away. As a result, variable gene...located near the origin will be over-represented compared to those further away. While constitutive promoters...gene expression during shifts in growth rate as compared to a constitutive promoter. To simulate the increase...

... changes when it goes from a more neutral pH compartment, ­­like the cytosol, to the higher pH of the ... to other pathways, it is standard practice to compare degradation rates between samples treated with ...the brightly-coloured Australian parrot. It’s comprised of two tandem fluorescent proteins: a relatively...Rosella’s excitation and emission spectra. Component Excitation Emission pH Range DsRed.T3 488, ...autophagy markers. It’s particularly useful for comparing bulk autophagy vs. mitophagy, the targeted autophagy...

...CasMINI plasmids Efficient CRISPR editing with a hypercompact Cas12f1 and engineered guide RNAs Yong-Sam Kim...result is a highly efficient Cas system with very compact genome editors and a gRNA down-sized by almost ...activity of Cas12f1 with these modified gRNAs is comparable to that of SpCas9. The group also confirmed that...Xue’s lab also uses paired pegRNAs targeting complementary DNA strands to program large genomic deletions...enzyme with a fully active Cas9 enzyme. PE-Cas9 comprises an active Cas9 fused to the reverse transcriptase...

...available in rAAVs!   Figure 1: Diagram of AAV components      Cargo capacity Empty rAAV has a cargo...conventional single-stranded AAV (ssAAV) and a self-complementary AAV (scAAV) vector. Full-length ssAAV vector...2.5Kb) and you need expression in a hurry, self-complementary AAV (scAAV) may be an option (McCarty et al....take the next step – selecting your AAV and AAV components (serotype and promoter) for use in your experiment... E. Monahan, and Richard J. Samulski. "Self-complementary recombinant adeno-associated virus (scAAV) vectors...

...below) Because off-target sequences have less complementarity to the gRNA, they have a higher propensity ...SpCas9-HF1 generated fewer off-target cuts when compared to WT SpCas9 across a variety of genomic sites...M694A/Q695A/H698A). This new variant displays comparable on-target activity and similar or decreased off-target...off-target activity across the sites tested when compared to eSpCas9(1.1) and SpCas9-HF1. The HypaCas9 plasmids... Hiroshi, et al. "Crystal structure of Cas9 in complex with guide RNA and target DNA." Cell 156.5 (2014...

... The destination vector contains sites with complementary overhangs that direct assembly of the final ...recognition site and permits DNA fragments with compatible overhangs to be efficiently assembled. Scientists...inverse orientation. This allows for multiple DNA components (promoters, genes, terminators, etc) to be assembled... reaction. For detailed Golden Gate protocols, complete with helpful tips and tricks, see The Sainsbury...system is an incredibly powerful tool for cloning complicated constructs in a single, high-efficiency step....

... don't do anything. Viruses are complicated and they perform complex biological tasks, both of which ...undergo DNA rearrangements. Depending on the composition of the viral vector and the repeat sequences ...perform a diagnostic restriction enzyme digest and compare it to some reliable controls. 2. Viral stocks may...ensuring viral production is to perform PCR against components of the viral vector using the harvested viral...

...benefits and drawbacks of different methods (for comprehensive protocols for all of the methods discussed here...quantified using qPCR primers targeting specific viral components such as LTRs, gag, WPRE, antibiotic resistance-genes...genomic DNA isolated, and primers targetting viral components or the transgene itself are used to quantify ...critical that standard conditions be used when comparing different batches of virus. In addition, some ...PMID: 19300443. 2. Geraerts, Martine, et al. "Comparison of lentiviral vector titration methods."BMC biotechnology...

...Revolution Bioengineering.  Last year we started a company. Revolution Bioengineering is two responsible adults...attend conferences makes it very challenging to compete in today’s tough academic or industry job market... this technology and angry at the agriculture companies that produce GMO food plants. When a press piece... – although we didn’t figure out how we would accomplish the color change until a few months later. RevBio... of our business, because we are building this company, and that’s what we want it to do. It’s exhilarating...

...expression baseline. Copy number is then determined by comparing results to a standard curve. For CNV assays, it...gives an absolute value of DNA concentration. By comparing the number of positive droplets for the gene of... through concentration values, rather than by comparison to a standard curve, and most results can be ...determination to move forward with your experiment?  Complexity of the gene of interest Some gene sequences ... S., Hollox, E. J., & Boon Peng, H. (2015). A comparison of assays for accurate copy number measurement...

...plasmids, they are generally electroporated into electrocompetent cells to ensure the highest possible transformation...use for your transformation(s). The amount of competent cells you need total and per transformation may...liquid culture, all of the bacteria can grow and compete in the same container, making it easier for a smaller...be its own colony, and will not be in direct competition with other colonies containing smaller, recombined...used for libraries that are similar in size and composition as examples. Table 1 provides an overview of ...

... pooled CRISPR library is a complex mixture. Pooled libraries are composed of many different gRNA-containing... and versatility. CRISPR/Cas9 consists of two components: the non-specific endonuclease (Cas9) and a single...any genomic locus, provided the target is unique compared to the rest of the genome and located immediately...distribution is analyzed in the drug-resistant population compared to a non-treated control group. In this scenario...

...multiplex (Thellwell, 2000). See Table 1 for a comparison of features of several commonly used chemistries...have pipetting errors on the edges of the plate. Compare the CT values within each set of triplicates to...reaction, each target in the reaction should be compared to an individually run sample when developing ...value representing the fold change of each sample compared to your control. But as we said previously, just... See Table 2, (adapted from Wong, 2018) for a comparison of different analysis methods.   Method ...defined log-linear phase   Table 2: A comparison of different qPCR analysis methods. Table adapted...

...feel comfortable speaking on topics that would compliment the STEM curriculum. As it happened, I’d met ...simplistic and I would insult the students. Too complicated and I would quickly lose their interest. I won...careful to keep it relatively simple. I honed the complexity of the talk by running it by the teachers and... For CRISPR, I focused on the fact that, like computers and the internet before it, genome engineering...posts Resources on Addgene.org Check out our comprehensive CRISPR guide Find CRISPR plasmids and resources...

...one such technique and has been used to produce complex fluorescent reporter constructs. These fluorescent...gene-expression sequences, and more. By combining these components, one can generate constructs to visualize cell...terminus or the 3’ terminus of the ligation product. Comparison to other high throughput cloning strategies There...modules, because neighboring fragments require compatible cohesive ends Engler et al., 2008 Gibson Cloning...facilitate the construction of plasmids with many components. The creators of the MXS-chaining method have...

... takes 1-1.5 hours to complete, while this syringe-based method can be completed in 20 to 30 minutes. ...PCR buffer, where DNA or RNA can be amplified. Compared to a commercial magnetic bead-based kit, this ...this did not reduce cloning efficiencies.  When compared to fresh columns, regenerated columns had similar... reduce the number of spin columns you need to complete your research. Do you know of other nucleic acid...

...will link biomolecules to the gel and add gel components to polymerize. Step 3: digest the sample to allow...labels you could use. But many ExM protocols are compatible with conventional antibodies, chemical dyes, ...and least flexible one. Tissue expansion is not compatible with in vivo applications and there’s just not...isotropic expansion depends on how well the gel components penetrate the sample, how well the gel polymerizes... valuable tool, especially when paired with complementary experimental approaches. Since it arrived on...