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...sufficient to give specificity and to also be compatible with a standard PCR reaction (see PCR Video)....design is similar, but we need to use the reverse complement to get PCR amplification. We can start similarly...the ORF). We now need to generate the reverse-complement of this sequence so that we can successfully ...amplify the ORF. You can generate the reverse-complement using existing software (a quick internet search... out Our Plasmid Reference Pages Deposit Your Completed Plasmids Find the Perfect Plasmid for Your Research...

...Zalatan JG, et al.'s recent paper, "Engineering Complex Synthetic Transcriptional Programs with CRISPR ... the eukaryotic cell for the execution of many complex cellular behaviors, such as differentiation or ...customizable single guide RNA (sgRNA) that bears complementarity to a 20bp target sequence (Qi et al. 2013)....appealing since the RNA scaffold modules are comparatively small allowing multiple sgRNA constructs to .... (2013). PubMed. Zalatan, J. G. Engineering Complex Synthetic Transcriptional Programs with CRISPR ...

...1); similar to an MRI or CT scanner. This is accomplished by focusing excitation light to a point in the... bleaching) to a single focal point. This is accomplished by using, for example, red light instead of ... photons of red light are needed to excite GFP compared to only one of blue. Only at the focal point of...coverslip and a sample in aqueous buffer it is completely reflected. This reflection occurs due to the ...immersed in. Although the excitation light is completely reflected, energy is propagated into the sample...cover samples of this size, those that can will completely image large tissues in a few hours. Although ...

...downregulate the expression of the major histocompatibility complex (MHC) class I proteins on their surfaces...immune response to the development of tumors is a complicated and dynamic phenomenon that is interestingly ...rear its head once the body’s immune system is compromised either due to age, infections, or other strenuous...focus on include: Cell heterogeneity Tumors are composed of a wide variety of cell types. This cell heterogeneity...successful tumor progression. A tumor’s genetic composition is constantly in flux and is dependent on the...

...and some home-made equipment. It was humble in comparison to the labs you’d find in Kendall square, but...Most of our equipment has been donated by local companies and BosLab has grown into an organization consisting... Timothy Stiles joined BosLab last year with a computer science degree and had zero pipetting experience...yeast to glow, produce synthetic scents, detect compounds in the environment, and openly publish protocols...the local community (including local garden and compost clubs) to participate in this study. How to get...

... other hand absorb best at 280 nm and organic compounds and chaotropic salts maximally absorb at 230 nm...SYBRGreen, are specific for double strand DNA (as compared to spectrophotometric methods that measure all... corresponding fluorescence. That way, you can compare the fluorescence of your sample against this curve...relative to the intensity in the ladder. It’s best to compare the band intensity of the fragment in the ladder...sugars under acidic conditions and forms a blue complex that can be quantified at 595 nm. This method is...

... precision gene editing. CRISPR-Cas9, the key component to enable efficient base editing and prime editing...unable to process its own array but is the key component to enable efficient base editing and prime editing... or any other kinds of CRISPR tools, without compromising their strategic advantages in gene editing, ...higher Cas9-independent off-target editing in comparison with single-site editing. Encouraged by the results...#186715) and hCtRNA_VT (#186716).   Fig. 2: Comparison of previous representative dual-gRNA architectures...

... potential binding affinity to major histocompatibility complexes (MHC class I and II).  There are multiple...of adverse reactions In silico assessment uses computer modeling and predictive algorithms to forecast...approach The in silico approach cannot replicate the complexity of antigen processing events that take place ...in-vitro models can be built that contain various components of the immune system. Figure 2: In-vitro...

...commonly used yeast by biotech and pharmaceutical companies is K. phaffii (formerly known as pichia). What...general research community. Phillips Petroleum Company first commercially developed K. phaffii in the ...Since then, multiple biotech and pharmaceutical companies (along with some research laboratories) have picked...of the uses outlined below. Biology They say comparison is the thief of joy, but I think it’s acceptable...10.3389/fmicb.2020.607028  Riley, R., et al., Comparative genomics of biotechnologically important yeasts...

...equivalent of crossing off days on the calendar after completing your writing goal, which can serve as positive...graph of which days of the week you most often completed your habit is useful for figuring out when to ...makes suggestions on how to make your writing less complex. While I don’t think you should accept all of the...jargon in your writing. While it’s not possible to completely avoid jargon, this tool can help you avoid overusing...collaborators. Zotero is a desktop based program that’s compatible with Mac, Windows, and Linux.  Mendeley and Zotero...

...need to be decided. Figure out what you need to accomplish to get to the next stage, and then adjourn. ... if any additional resources are needed. All-Company Meetings: As your organization grows, so will the...to address one point aloud. This keeps our all-company meetings short, informative, and pleasant. Email... positive peer pressure to make sure everyone completes their assigned items to keep the project moving...timely feedback on each other’s projects. For all-company meetings, monthly still works best. Figure out ...

...transcription to begin. In addition to arabinose, cAMP complexed with cAMP activator protein (CAP) can also stimulate...another way to activate gene expression, and two-component systems used in synthetic biology use light to... Addgene also has popular light-regulated two component systems designed by the Tabor lab. Which inducible...crossover. The induction mechanism must also be compatible with your experimental system. Leakiness If you...Region Plasmids 101: Cre-lox Light-Switchable Two-Component Systems for E. coli Browse other blog posts from...

...Cas9.  For a brief comparison of Cpf1 vs. Cas9, see the table below. Table 1. Comparing the Cas9 and Cpf1...simplified system for editing multiple targets compared to Cas9.  Read on to learn more about Cpf1 multiplexing...Cpf1 CRISPR Nucleases   Cas9 Cpf1 Components crRNAtracrRNACas9 crRNACpf1 Endonuclease size ...expression vector.  See the graph in figure 2 for a comparison of editing frequency resulting from transfection...

...Anti-FLAG frankenbody (FB) construction from the complementarity determining region of an anti-FLAG scFv and... variations in DR sequences. Figure 2: comparison of Cas 9 vs. Cas 12a expression cassettes when... of performing similarly to Cas9 screens in a compacted library. Overall, Cas12a’s ability to target multiple...CRISPR-Cas system for editing and gene activation, is composed of only 529 amino acids, less than half the size...or Cas12a. Despite its small size, CasMINI is comparable in efficacy to dCas12a systems in both gene activation...

...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...

... You can do more than just ‘sense’ biological compounds and reactions; you can quantify them with biosensors... how to use viruses to set up these systems. Components of a biosensor Some of the biosensors used in... that you are interested in. The most obvious component of a biosensor is the thing that you care about...or an electrolyte such as calcium. This varied component, which we will refer to as the analyte, needs ... fluorescence, pH change, heat, etc. The last component of the biosensor is the signal transducer which...

...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...

... long as their substrate is available. Table 1 compares some general advantages and disadvantages of each... proteins to be visualized simultaneously. Complementation systems for examining protein-protein interactions...binding interaction brings the two luciferase components together. A similar approach can test protein-protein...Luciferases can only produce light in the presence of a compatible substrate, which varies depending on the specific...luminescence from live cells. Luciferase variants with incompatible substrates can also allow multiplexing with ...

...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...

...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...

...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...

...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...

...you’ll want to ensure that your buffer components are compatible with your conjugate and conjugation chemistry...the assay. Where possible, it can be useful to compare recombinant antibodies to their parental hybridomas...majority of antibodies are still equally effective compared to those aliquots frozen immediately post-harvest...Chen E, Haidar JN, Srivastava A, Goldstein J. Comparison of predicted extinction coefficients of monoclonal...

... will apply to similar instruments from other companies, like the Implen NP80 or DeNovix DS-11, but some...lines in B-D represent the pure DNA spectra for comparison. Each of these contaminants will have a slightly...of a DNA sample, its absorbance strength is low compared to other reagents, making it more difficult to... in your DNA sample, you will need to use a complementary assay, such as a fluorescence based assay or...more consistent readings — just be sure it is compatible with your downstream applications and that you...

... 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...

...collaborations, and commitments to and proof of compliance with the grantor’s requirements as well as state...reports are submitted and project evaluations are completed. To enter and advance the profession, you can ...Professional Development Strategy to increase your competencies and skills in the grants profession. Your professional... and the candidate’s knowledge of the 8 core competencies through a multiple choice exam.  The bottom ...grant professionals and to build your skills and competencies. Becoming a grant professional is rewarding ...

...into a translated protein. As depicted in the accompanying cartoon, they have a multitude of uses including...TAP) once referred specifically to a combo tag comprised of a calmodulin binding peptide (CBP), a TEV cleavage...ProtA IgG-binding domains. TAP has since come to encompass several other tag combinations, though frequently...and Gly residues   This article is not a comprehensive guide to all tags, but rather a quick overview...Biotechnology Journal 2012 , (7): 620-634. Full Text A complete list of free handbooks for protein purification...