...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...

...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...