... desired mutation, then use those to PCR amplify the template plasmid. The parental, nonmutated DNA is...containing the desired mutations, to amplify both strands of a plasmid template. In addition to containing the...mutations, large deletions/insertions require complicated, often costly in vitro assembly methods. A new...nick repair. This method can work well, but poor template digestion can result in a high number of background...extension PCR is used to splice together two PCR amplicons based on sequence homology, and the subsequent...After being treated with DpnI to digest away the template plasmid, these PCR products are directly transformed...pitfall of REPLACR-mutagenesis is that you must PCR amplify the whole plasmid, which could introduce mutations...

...plasmid monomer from a plasmid sample, or if no monomers exist in your sample, you could try linearizing ...control when performing a diagnostic digest is a simple but effective method for detecting plasmid multimers...Bedbrook & Ausubel, 1976). In fact, if your plasmid sample has a mix of different-sized multimers, you may...corresponding to each type of multimer present in your sample. In these instances, please note that the supercoiled...agarose gel). If the presence of a multimer in your sample remains unclear after visualizing it on a gel, ...perform whole plasmid sequencing of our plasmid samples during our quality control process. Since MiSeq...topologies and multimers present within a plasmid sample. Troubleshooting Tips   Figure 3. Summary...

...are having difficulty amplifying your target sequence, or if you want to amplify sequences from a large..., but if you are having difficulties in amplifying a template, particularly from a large genome, you can...Assembly, Golden Gate etc.), and try to find the simplest way to do it (i.e. avoid assembling too many fragments...Design primers that overlap at least 24 bp with the template and if the sequence has high GC or AT content,...Remember to quantify the DNA concentration of all the samples by gel or by nanodrop to assemble the  fragments...correct insert Sequence the regions that have been amplified by PCR, putting special attention in the areas...plasmid. Many vectors contain sequences that are complementary to standard sequencing primers. If not, design...

...“Vector Type Other” column. For example, you may enter “donor template vector”. Cloning Method Choose...plasmid deposit process can be broken down into four simple steps: Send plasmid information to Addgene Material... explain how to use our deposit spreadsheet to complete Step 1. Steps 2 and 3 are usually very easy — ...the Deposit Spreadsheet is the quickest option. Simply download the spreadsheet and copy and paste your...any functional consequences of that change. For example, “Mutated aspartate 123 to Lysine (D123K), lowers...contact Addgene. We may require that you send your samples to Addgene pre-transformed in this strain. ...and sending the deposit spreadsheet Figure 2: Example of the deposit spreadsheet showing the first eight...

...reducing editing from ~30% in control samples to 0-10% in Acr samples. Acr proteins can also be used to prevent...assembly of the crRNA-Cas complex (Zhu et al., 2019). Inhibiting DNA cleavage Example: AcrIIC1 binds to the... of discovery (Bondy-Denomy et al., 2018). For example, AcrIIA4 mentioned above indicates that it’s the...activity one of three ways: Inhibit DNA binding Example:  AcrIIA4 blocks Cas9’s interaction with the PAM...site (Harrington et al., 2017). Inhibit crRNA loadingExample: AcrIIC2 interactions with Cas9 interferes ...and spatial control of CRISPR-Cas activity. For example, fusing AcrA4II to the photo-sensitive LOV2 domain...selectable markers in engineering viruses. For example, an Acr gene was used to replace genes in the difficult-to-engineer...

...high and above what would be expected in patient samples (e.g.: blood, urine). Adapting toehold switches... dozen versions of the toehold switches and amplification primers. This helped make sure that we had a...up clear tasks for each member of the team to accomplish so that many elements of the project were able...involved definitely contributed to our ability to complete the project on such a short timescale. Our article...pathogen’s genome as unique barcodes to identify samples containing the virus. Of the 48 sensors, targeting...this includes primer directed isothermal RNA amplification, new toehold switch designs, CRISPR/Cas9-mediated... diagnostic results.  Our approach now detects samples at clinically relevant concentrations (1-3 fM; ...

...use this tool to detect a BRAF gene edit, simply PCR-amplify the BRAF locus from the edited cells (the...gRNA-transduced samples (data not shown). Pro-Tips: 1.Designing primers against genomic DNA to amplify the gRNA...and GFP-edited amplification products (BRAF-GFP duplex) and from GFP-edited amplification products (GFP-GFP... wanted to inactivate the gene BRAF (a kinase implicated in several human cancers) in A549 cells (a human...should contain the edited BRAF sequence), and PCR-amplify the BRAF locus from control cells (the PCR product...genome browser and Primer3 to design primers that amplified a 500-600 bp product. The primers I designed (...gel that highlights the important results (for example, if you needed to make a figure with the data)....

...essays in the journals Nature and Science, for example). This blog post is about how to get the most ...requires that all of the experiments have been completed. Therefore, to be ready to apply for a Group Leader... questions like: What figures are missing? For example, are additional controls necessary? As you build...Discussions can reveal mutual scientific interests, complementary perspectives, a shared communication style. ... week planning an experiment, even a relatively simple experiment. However, by taking this time, you can...master. While the calculus of these decisions is complex, here are a few guidelines to help you evaluate...result? Is there a tractable question or are you simply describing a phenomenon? Exploration can be important...

...relatively easily synthesized, must be produced using complex mixtures derived from cells or using live cells...expression consists of transfecting cells with your DNA template of choice and allowing these cells to transcribe...systems Insect cells can also be used to produce complex eukaryotic proteins with the correct post-translational.... These systems enable high expression of very complex, glycosylated proteins that cannot be produced ...expression. Protein expression in bacteria is quite simple; DNA coding for your protein of interest is inserted...pET, pRSET, Gateway pDEST, and pBAD vectors for example. Protein expression from each of these vectors ... expression systems, including the majority of complex post-translational modifications. Extraction and...

... DNA and processed the mixed sample in our pipeline (see Figure 2, sample 2). When we blindly analyzed...these known impurities, as was the case for clean sample AAV-44362. In addition to these expected hits, ...to “random” genes. Do these hits always mean the sample is contaminated? No - what really matters is the...of hits to the same sequence (gene). In a clean sample, there are typically fewer than 10 hits to a given...>100 hits - immediately raises a red flag. For example, AAV-68544 contained 220 hits to the CHRM4 gene...proceed to de novo assembly of the unmapped reads, employing our bioinformatics software to assemble the reads... the contaminant we mixed into the original AAV sample at 5%. Detecting 5% contamination is a good start...

...homologous recombination. The concept is simple: deliver a template with a modified version of the targeted...homologous recombination using a repair template. These templates can include loxP sites to create conditional... sequence to the cell which will recombine the template with the endogenous gene. Here, we'll describe...homology is typical for targeting constructs. In the example shown in Figure 1, large sequences corresponding...is expressed (knocked in). As seen in the above example, you can remove a floxed resistance gene using ...plasmids described in the preceding sections are simple ways of knocking out a non-essential gene of interest...functions normally because one of its exons has simply been replaced with the same sequence flanked by...

...protein fusion. CRISPR genome editing can help accomplish this goal. The power of this system lies in the...individually generate gRNAs and homology arm repair templates containing the tag for each insertion. Also, it..., as well as the 5’ and 3’ homology arm repair template (Fig. 1, Step 1). There is no need for PCR or ...the second site accepts the homology arm repair template. SapTrap includes a prebuilt donor plasmid library...be used to generate functionally unique repair templates. A final ligation reaction correctly assembles...genetic locus, the toolkit also offers repair templates for tagging proteins in a tissue specific manner... CRISPR/Cas Plasmids - Protein Tagging. Beyond simply monitoring protein localization and activity, CRISPR...

... useful they are implemented in most materials that have been engineered. For example, Boole’s logic shows...BioBricks Foundation to distribute the physical DNA samples, for when that's the preferred mode of receiving.... What the BioBricks Foundation is seeking to accomplish is to make the engineering of biology much easier...Integrase Logic (BIL) gates collection of parts implements the basic logical functions, which we know and..., computers, and now through BIL gates we've implemented them in DNA. We thought these were so much fun...including growth or fluorescence. There are a few examples of engineered RNA switches, but we've not yet ...incredible hidden story here. The parts we are releasing simply plug into this existing economic activity. People...

...valuable to scientists, there are some potential complications that must be considered when using this method...alternative approach, which avoids many of these complications, is to directly deliver a ribonucleoprotein ...ribonucleoprotein (RNP), consisting of the Cas9 protein in complex with a targeting gRNA, to your cells of interest. Cas9...CRISPR experiment is the generation of the RNP complex.  While one option is to purchase the Cas9 protein... nuclear membrane, allowing for entry of a DNA template. Other techniques, such as lipid-mediated transfection...specific cell types (Rouet et al. 2018). This was accomplished using a Cas9 mutant (Cas9M1C/C80S) that harbors...been an area of intense study. Two methods to accomplish this are Polyethylene Glycol (PEG) mediated transfection...

...little intimidating. Too simplistic and I would insult the students. Too complicated and I would quickly lose...I was careful to keep it relatively simple. I honed the complexity of the talk by running it by the teachers...feel comfortable speaking on topics that would compliment the STEM curriculum. As it happened, I’d met ...will likely change for next time is giving fewer examples and think of a way to make this section more interactive...present to middle schoolers, is to provide current examples. When I asked Joanne Marks, one of the 8th grade...extension to our curriculum and offered engaging examples of how understanding genetics is so timely.” Genome...hard-pressed to maintain the pace of CRISPR. Two of the examples I used in my presentation had only become public...

...multiple Cas proteins to form the Cascade targeting complex is a hallmark of Type I CRISPR-Cas systems, in ...in contrast to single-protein effectors employed by Type II systems (such as CRISPR-Cas9). Unlike in regular...nuclease activity. Instead, it forms a novel co-complex with the transposition protein TniQ for targeting... by the system. Guided by the crRNA, QCascade complexes survey the genome and bind target DNA sequences...pairing. Separately, TnsABC forms a transposase complex with the mini-transposon, excises it from the donor... a very similar manner to Type I-F, but instead employ a Cas12k protein instead of three Cas proteins ...in single-plasmid INTEGRATE (pSPIN), which offer simple transformations for smaller cargos, and the ability...

...removing excess dye from the sample, which is particularly important for samples where washing is difficult...reason is something called “the photon budget”: each sample has a limited number of fluorophores and each fluorophore...of information one can extract from a biological sample is wholly dependent on the photon budget and pushing...fluorescence microscopy often requires more photons. For example, moving from transient overexpression of protein... desperate to extract a few more photons from a sample. Chemical fluorophores can be substantially brighter...labeling chemistry easier and more functional in complex biological environments such as live cells and ...brightness of the resulting conjugate, and the complexity of the system. A particularly desirable property...

...credit for what we have accomplished. Starting with a recap of accomplishments, from learning new techniques...tangible research and career goals, and celebrate accomplishments. These techniques are laid out in Vincent et...DePace’s yearly planning meetings revolve around two simple worksheets, each of which is filled out separately...conversation. Goals and Planning Worksheet: Lists accomplishments, research/professional goals and feedback. ...including major project goals and effort needed to complete them. These documents dictate the subject matter...concrete plan in hand, trainees are motivated to complete their work rather than overwhelmed or confused...Extracurricular Activities for a Strong Science Career Path 7 Simple Strategies to Resolve Conflict With Difficult Supervisors...

...desired tags have been integrated. Simply design your amplification primers with the desired targeting...cerevisiae and S. pombe. The protocol is surprisingly simple, requiring only a PCR product containing the modifying...markers. These are especially useful in protein complex studies where tagging of multiple protein products...for easy capture and detection of proteins and complexes, without the artifacts sometimes associated with...Formosa, at the University of Utah, has built a complete collection of yeast tagging modules with each ...ideal for tandem affinity purification of protein complexes. John Pringle and Jürg Bähler have deposited a...GST, or GFP). Longtine et al.(3) describe a complimentary set of plasmids for use in S. cerevisiae, with...

...needed to transform the raw sequencing data into a complete plasmid sequence that would be ready for our QC...containers are they are designed to ‘contain,’ in a complete package, all of the pieces of software that depend...can be easily shared with others without any complicated setup. We created, tested, and debugged each ...bioinformatics workflow in Nextflow Establishing a complete pipeline however, required more than just identifying...executed on Linux, we created our first process by simply copying and pasting pieces from our existing script...from the first process to the second process. For example, our bbduk process created trimmed FASTQ files ...sure we understood the concept of channels and implemented the input declaration correctly, the script block...