We narrowed to 928 results for: Mpl

...cerevisiae yes - This can complement ura4- S. pombe, but the complementation is weak. yes   LYS2 L-lysine...vectors? Yeast are eukaryotes and thus contain complex internal cell structures similar to those of plants...mechanism of homologous recombination to facilitate simple gene replacement/mutation. Yeast expression plasmids... including a copy of a functional gene which complements the host’s auxotrophy. When grown on media NOT...LEU2 L-leucine no S. cerevisiae yes - This can complement leu1- S. pombe, but multiple copies are required...drug selection methods feasible in yeast as a complement or alternative to using auxotrophic markers. ...antibiotic resistance cassettes is most commonly employed, with the KanMX being the most prevalent due to...

... the cell with a mutant template and fooling the cell into using that template for repair. By using these...the two ends back together or using a homologous template provided by the sister chromosome. As cells take...especially well-suited for use in stem cells. (See the complete protocol at StemBook). Addgene: How are you now...diseases. We are knocking out genes that have been implicated in disease or, in cases where particular variants...disease process. In the Cell Stem Cell paper, for example, we generated hepatocytes to examine the effect...much all investigators. Our kit makes it extremely simple to design TALENs. It only takes two to three days...less than a month's time. Addgene: What are the implications of this kit in terms of understanding human ...

...detected with tyramide signal amplification. Tyramide signal amplification occurs when HRP converts a fluorescently...used at a time. Samples can be sequentially stained with two HRP RANbodies if the sample is stripped. HRP-RANbodies...Tyramide signal amplification of HRP Staining cultured cells and tissue sections Amplifying weak signals...process that’s difficult for individual labs to complete. The nanobody based RANbody platform from the ... lower background and better penetration of the sample due to the smaller size of RANbodies compared to...

...but what if we had a simple tool that allowed us to rapidly decode the complexity? The CombiGEM-CRISPR...was contributed by guest blogger Alan Wong. The complexity of biological systems can hinder our attempts...of providing an easy-to-use tool to analyze the complex combinatorial genetic networks underlying your ...experiments. CombiGEM-CRISPR: The marriage of two simple platforms CRISPR has revolutionized how we decode...mapping the combinatorial genetics that underlie complex regulation in biological systems. The CombiGEM ...new technologies to interrogate and understand complex biological systems. He can be contacted at aslw...transcriptional activation by an engineered CRISPR-Cas9 complex." Nature (2014). PubMed PMID: 25494202. PubMed ...

...QCascade complex surveys the genome and binds its target DNA sequence. The transposase complex binds the...really depends on where a transposon lands. For example, in bacteria, transposons can move antibiotic resistance...is left behind. When the transposon-transposase complex finds its target site, it integrates, which produces...TEs You can think of autonomous transposons as “complete transposons” since they encode the protein they...selecting a transposon for a mutagenesis screen. For example, piggyBac and Tol2 are best for screening promoters...DNA-targeting module with the crRNA, 3) a transposase complex made up by three transposase proteins (TnsA, TnsB...

... opportunities for us all.  For example, implementation and compliance for the FLSA changes, including...turn. The first is the passing and upcoming implementation of the Federal Labor Standards Act (FLSA) which...remain mixed in their opinions on the subject – for example, Yale graduate students filed for union elections...union dues. Time will tell the extent of the implications of this ruling and whether graduate students...finding money in already tight lab budgets and even simply being able to categorize all postdocs at institutions...challenging, but will be aided by the development and implementation of new strategies designed to cope with current...

...structure of the hairpin-DNA complex is thought to destabilize the transcription complex, initiating cleavage ...transcript. Rho-dependent terminators are not usually employed in plasmid-based expression systems, so these ...100% efficient at halting transcription of the template and initiating the desired cleavage event, although...polymerase terminates differently. Polymerase III, for example, relies on a specific sequence and RNA secondary...termination factor dependent, Polymerases I and II employ different mechanisms to terminate transcription...mechanism, whereas Polymerase II termination is more complex and involves two RNA polymerase-associated proteins... care (4). For this reason, viral vectors often employ other non-poly(A) transcript-stabilizing and nuclear...

...transfer plasmid. The good news is there’s a few simple ways to deal with this issue. One way is to grow...helps me figure out if I have enough plasmid to complete my experiment. AAV is usually the limiting reagent...concentrate the purified virus. This process can be completed in one long day, or the virus can be stored at...particles that are present in an AAV prep. AAV samples are first DNaseI digested to remove any residual...technology or a TaqMan primer/probe set can be used and samples are quantified by comparing to an AAV standard ... plasmid. This whole process takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR...AAV, remember to account for the dilution of the sample for the DNAseI digest. Use good sterile technique...

...inexpensive and can be 5’-phosphorylated in house using a simple PNK reaction or can be purchased 5’-modified from...probably while it is being synthesized) can form a complex with an ssDNA guide. Therefore, researchers must...inject cells with in vitro preassembled Cas9/gRNA complexes or with guide RNA- and Cas9-expression plasmids... an optimal distance from the target site. For example, NGG may not be present in AT-rich regions of the...same method used by Gao et al, 2016 (1). View the Complete and Up-to-Date Results of Pooran's NgAgo Survey..., leaving a small amount of active NgAgo/ssDNA complex in the cell. As proposed in the original article...protein molecules manages to form a functional complex with ssDNA leading to inefficient downstream editing...

...about a month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction...double-stranded break. HDR copies the sequence from a repair template with flanking sequence homology for error-free...systems, and NHEJ will be favored even if a repair template is present. HDR also presents a cloning dilemma...required by MMEJ can easily be added via PCR amplification. Given the inefficiency of HDR for knock-ins...strategy looks very similar to HDR, but it is accomplished using much smaller regions of homology, which...strategy looks very similar to HDR, but it is completed using much shorter sequence homologies. Abbreviated...Select for puromycin resistant cells Step 5: PCR amplify and sequence the locus to verify correct GFP-puro...

...over previous genome editing techniques is its simplicity and versatility. CRISPR/Cas9 consists of two ...jar of jellybeans, a pooled CRISPR library is a complex mixture. Pooled libraries are composed of many ...Pooled lentiviral CRISPR libraries (referred to simply as “CRISPR libraries”) consist of a heterogeneous...for experimental use. Thus, the first step is to amplify your library to a concentration that is sufficient...next-generation sequencing to check the quality of your amplification. If you've obtained a ready-to-use lentiviral...sorting and screened for a particular phenotype. For example, mutant cells can be used in drug screens to identify.... Figure 2: The CRISPR library must be amplified (A) to be used to generate lentivirus (B). Cas9...

...carry their emission peaks within their names, for example, BV421 and RB545. Fluorophores can also come in...recognize a couple of fluorophores from Figure 1. For example, the chart tells us that PE and PE-Cy7 are "very...en-us/resources/bd-spectrum-viewer.   Another example APC is a "bright" fluorophore when excited by the... of neighboring colors can and do overlap. For example, APC overlaps noticeably with both BV605 and Alexa...antibody-conjugated labeling. Even though the two compounds are completely different in nature (eGFP being a protein and...build your first panel! Designing a Panel In this example, I will describe a basic panel (Table 2, Figure...much higher than CD45 expression, this could, for example, point at issues with the double-expression vector...

...years ago. Teaching by example No examination of the state of science would be complete without the inclusion...attempts to answer it in five paragraphs, with examples. If that sounds ambitious, that’s because it is...non-scientist friends and family is Part Five, called simply “Bad Science.” Zimmer kicks off the section saying...provides several. In Part Three of the book, called simply “Old Science,” he describes the now infamous tale...Four, “New Science,” Zimmer tackles more recent examples of scientific advances, and posits the way science... much less familiar with as a biologist--as an example of “big science,” a massive, expensive project ...CRISPR of course address some of the ethical implications of using these technologies, but also the rapidity...

...referred to as “complex retroviruses ” (Cullen, 1992) because of their more complicated genomes. Here, ... we will mostly use the gamma-retroviruses as a simple spokesmodel for the whole family; however, you ...beginning to realize that the situation is already complex enough that a deep understanding of retroviral ...polymerase gene may seem like a mystery. But it’s simple. The RT protein is made by a separate set of instructions... a tool is done, and the transgenic genome will simply express from whatever promoters were included in...Mechanism of action of regulatory proteins encoded by complex retroviruses. Microbiological Reviews, 56(3), 375...Initiation of synthesis with 70 S viral RNA as template. Journal of Molecular Biology, 79(1), 163–183....

... will guide the cell to use the donor DNA as a template for repair, which will result in the incorporation... knock-in! So how does the donor get used as a template for repair? Read on to find out! Homology-directed...basis of the HDR pathway relies on repair from a template molecule, which is usually an endogenously available...and uses the ends of the ssDNA to find a repair template. The highest HDR efficiency is observed when inserts...making the cut off essentially as part of the templated DNA. Check out our blog post on increasing HDR...To make sure your donor molecule is used as the template, you need to incorporate homologous sequence to...sister chromatid is likely to exist as a repair template. To maximize HDR events, ensure cells are actively...

... OriP and induces amplification of the episomes concurrently with DNA amplification in the host cell. ...blood sample, in the best-case scenario [7]. In the clinical setting where patient blood samples are limited...especially if used to make clinical grade hiPSCs. For example, the CytoTune™-iPS 2.1 Sendai Reprogramming Kit...persistence of residual viral material [6]. The complete removal of the residual viral particles and establishment... in the presence of these two factors are not completely understood, the addition of an shRNA for P53 ...grade hiPSCs from easily accessible patient blood samples. All pCXLE vectors listed below in Table 1 are ...

...surface) of the leaf. This is a simple and easy technique to implement. Success might vary between experimenters...tobacco, Agrobacterium infiltration into leaves is a simple and efficient way to overexpress a gene and evaluate...your experiment (de Ruijter et al., 2003). For example, we did our initial transformations with the GUS... on transformation success. Some reporter gene examples are below. The GUS gene (β-glucuronidase) with...temporal reporters to measure promoter activity, for example. Useful positive controls could include: pSB96 ...expression from Agrobacterium, but not the plant. For example, we have seen strong expression of the GUS gene... Li, Jian-Feng, et al. "The FAST technique: a simplified Agrobacterium-based transformation method for...

...chalk talk? On the surface, a chalk talk is quite simple. It's you, a dry erase board, some markers (chalk...question, not a vague goal or a "what" question. For example, "we want a better understanding of the Z pathway...about the time and resources required to feasibly accomplish the aims and clarify what type of aim you are...scientists outside your field, and interesting. For example, “there is a significant need for healthy livers...explain why it’s significant and innovative. For example, "while it is possible to grow liver stem cells...exist to solve the problem. What will you have accomplished in 5-10 years? You need to be able to describe...learned and/or now be possible to do? Research accomplishments are remembered for the problem solved, not ...

...in which the two guiding principles have been implemented by providing support for connectivity to external...Without such references the record of research is incomplete, making it difficult or impossible to reproduce...Github and Gitlab, and science-specific tools, e.g. sample management apps, colony management software, and...the record of research captured in the ELN is incomplete. Thus, to support reproducibility an ELN should...provide a direct boost to reproducibility. For example, it’s possible to import Word documents into RSpace...possible to link between various documents. A second example is the integration between RSpace and Slack, illustrated...subsequently reproduce the research. A third, powerful example is the integration with protocols.io, the protocol...

...for implementation was set for Dec 1 2016. In order to be exempted from the need to pay employees overtime...submitted letters to the Department of Labor (for example here and here) pushing for exemptions for postdocs...institutions doing? Institutions are now preparing to comply with the changes to the law on Dec 1st, according...centrally administered, which leads to many complications, including the inability of some institutions...that they are raising salaries as their website implies. These 5 organizations, according to the NSF data...they're thinking about applying to, are doing - for example, your benefits may be changing. Please also feel...wide consensus of academic stakeholders. There was ample opportunity to accept these recommendations, and...