...us to the key question: what is the best way to compare the photostability of different fluorescent proteins...measurements for different fluorescent proteins and by comparing how the fluorescence intensity changes over time...photostability of the fluorescent proteins can be directly compared. An example of a photostability measurement of...fluorescent protein can be identified by a head-to-head comparison of several fluorescent proteins of the same spectral.... In figure 1 we provide an example of such a comparison between cyan fluorescent proteins using widefield... power, the photostability of the variants is comparable (lower panel of figure 1). To avoid changes in...

...specific chromosome. This image represents the composite of fluorescent measurements from red, blue, and...modified GFP-dCas9 and the gRNA scaffold to enhance complex assembly, thereby decreasing the amount of background... labeling efficiency was similar to that of a comparable FISH method. Importantly, they did not observe...with the previously established physical map. In comparing pairs of targets ~2 Mbp apart, they noticed that...that they could evaluate the degree of chromatin compaction even for this small distance! To the authors’...CRISPRainbow generates 6 unique colors and a white composite. gRNAs with two identical hairpin binding domains...

...specific components that are key to the cloning reaction. Understanding the specific components is essential...take up your insert. But exactly how does ccdB accomplish this? Let’s start with a brief history of the...system contains ccdB. A successful insertion will completely replace ccdB with the investigator’s insert of...interest.   CcdB resistant E. coli strains complete the system But ccdB alone isn’t the only key to...protein involves poisoning of DNA-topoisomerase II complexes.” J Mol Biol. 1992 Aug 5;226(3):735-45. PubMed...

... and trolls. People don’t like to connect with complete strangers as it borders on my first rule of networking...’m asked a lot about requesting connections to complete strangers for informational interviews. Many have...option is in the “More…” menu. You can also follow companies and organizations to see their news and job postings...interviews.  Pro tip: You can learn a lot from the company LinkedIn page. This can include news, inclusion...It’s also a great way to keep track of past accomplishments. Follow all the rules of good resume writing...writing. Include examples of concrete accomplishments in both the technical and “plus skills” areas. Include...

... do as genetic circuits become larger and more complex? Voigt: If you look at all the products of biology... on genes at different times. It requires more complex synthetic networks. Addgene: What hurdles did you... perspective and you can layer them for more complicated functions. Those things are easy with CRISPR..... There haven’t been enough genetic parts for complexity. We have characterized almost 600 terminators...of a cell. The objective is to give people the complete toolbox for taking on very large genetic engineering...just using what biology has given us. Wood is a complicated material and we can access it, but we are stuck...

...science and collaboration. The field already has comprehensive reference pages and curated databases for scientists... (CeNGEN) The CeNGEN goal is to establish a comprehensive gene expression atlas of every C.elegans  neuron...that is creating the “first virtual organism in a computer.” This project has produced various resources ...four-dimensional contrast interference (DIC) microscopy and computer imaging processing. The combination of these methods...methods provides unique opportunities to develop computational methods to better understand animal development...Hammarlund, Marc, et al. "The CeNGEN Project: The Complete Gene Expression Map of an Entire Nervous System...

...blood clotting, and as we all know, a structural component of bones and teeth. It is an enzyme co-factor,..., a neuron subtype) and specific subcellular compartments for measurements. All of the sensors discussed...calcium concentrations in different subcellular compartments can vary greatly. Additionally, the calcium ...susceptible to artifacts and pH interference, and comparison between experiments can be more difficult due...GCaMP6 only) or mammalian cell expression, and compatible with constitutive or Cre-dependent expression...Genetically encoded calcium indicators to probe complex brain circuit dynamics in vivo. Neuroscience Research...

... to stop functioning properly or be something completely different than what was originally intended. ...tags, promoters, and other critical functional components. Fun fact! Some older plasmids in our collection...each of their plasmids, from descriptions of key components of the plasmids to partial, full, or annotated...Addgene receives deposited plasmids, they undergo complete plasmid sequencing using an in-house Illumina ...Addgene. The assembled plasmid sequence is then compared to the information provided by the depositor. ... sequence is not available, QC scientists will compare the NGS results to the plasmid information, other...

...that allow their host organisms to thrive in competitive environments. The genes encoding these functions...triumph over your neighbors is to eliminate the competition, and Penicillium isn’t the only organism that... are also less aggressive ways to survive in competitive environments — like by living where no one else...to be active only in the presence of the target compound. These systems are a gold mine of parts for synthetic...Shareck et al., 2004). Plasmid categorization is complex Plasmid classification has shifted over the years...necessarily reflect genetic relationships or plasmid incompatibility. Plasmids may also have more than one function...

...significant amount of flexibility compared to a strict single-compatibility sequence like NGG. Nonetheless...NG, a rationally engineered Cas9 with NG PAM compatibility, as the name implies (Nishimasu et. al, 2018...know and love about Cas9, but with broader PAM compatibility. This alternative PAM requirement adds a significant...PAMs and is capable of editing at frequencies comparable to SpCas9. SpRY can also edit NYN PAMs, but with... Ai C, Hu J. In-depth assessment of the PAM compatibility and editing activities of Cas9 variants. Nucleic...

...remodeling. Chromatin is a complex of DNA and histone proteins that compacts cellular DNA into chromosomes...in the compacted region and results in a loss of gene expression. In this way, genes in compacted regions...and tightly bound DNA is to histones, the more compacted it becomes. This prevents the cellular machinery...have huge impacts in the way we study and treat complex or multifactorial diseases, enabling faster diagnosis...

...information directly into the file. Email the completed spreadsheet to [email protected] along with ...used to knockdown expression of mRNAs with complementarity to the shRNA sequence. Empty backbone A plasmid...plasmid data in the pages described below. You can complete these pages sequentially by entering the appropriate...the links at the top of the page. Figure 11: A completed plasmid page for review before submission. Once... right of each data entry page. Once you have completed the data entry process for all of the plasmids...Figure 13: Click the "Submit Deposit" button to complete the data entry. Clicking the "Submit Deposit" ...third party rights holders. Once this process is complete, you will receive a Deposit Confirmation email...

...bacterial strains, like NEB Stable. NEB Stable competent cells lack RecA, a protein that aids in the homologous...helps me figure out if I have enough plasmid to complete my experiment. AAV is usually the limiting reagent...produce more virus because I don’t have enough AAV. Compared to the Addgene AAV Production protocol, I take...concentrate the purified virus. This process can be completed in one long day, or the virus can be stored at...set can be used and samples are quantified by comparing to an AAV standard curve, which is generated from... plasmid. This whole process takes ~3 hours to complete: 1 hours hands-on time and 2 hours for the qPCR...

...while also ensuring that my work gets completed? I want my company to pay for me to attend a conference... How can I negotiate my starting date with the company so they know I’m excited while still being able...want to learn more about a different area of the company. How can I negotiate with my manager to gain experience...conference. How can I negotiate to show the company that investing in me is also beneficial to them? I have an...but I need help from another person/team at the company. How do I negotiate with the person/team for help...

...about a month and a half, without the need for complicated cloning of homology arms. MMEJ: An Introduction...HDR and NHEJ, please see the linked blog posts. Compared to its counterparts NHEJ and HDR, MMEJ doesn’t...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...efficiency of PITCh-based genomic insertion would compare to CRISPR sticky-end insertion using the nuclease...Genome-Editing Based Approach for Mapping Multiprotein Complexes in Human Cells.” Cell Rep. 13: 621-633. PMID: ...

...know that this process only became even more complicated with COVID-19 and standards for hybrid, virtual...without the financial and logistical stress that accompanies traveling for campus visits. This is definitely...where the PhD application cycle becomes much more complex. “How do I reach out to potential mentors?” “Where...very valid questions, and often make an already complicated process even more difficult. I’ve learned that...to be incredibly useful, as it gives a more comprehensive idea of that advisor’s research interests which... for the interview. Having notes close to your computer with things you want to highlight in the interview...

...These insights are hard to appreciate without computational support. Indeed, rational sgRNA design is not...experimental applications in CRISPR engineering. When compared to that collective utility, it’s easy to fault...tool unique. The CRISPR Software Matchmaker is composed of these features and enables you to select the... based on their comfort level with operating a computer.  Examples: “offline”, “online”, “CLI”, and “GUI...few offline tools available mostly work on any computer but generally require working knowledge of the... power of each tool they are interested in by comparing their past results to the designs suggested by...pattern at any edit distance. It won’t, however, compute any score other than the edit distance. Added functionality...

...just one or two recombination events, homemade competent cells of 107 CFU/μg are perfectly fine, yet performing...performing multiple modifications will require ultracompetent (109 CFU/μg) cells. Primer design Using specific...insertions, deletions, and point mutagenesis to complex assembly of multiple genes. Primer design for each...the forward primer by adding a short sequence complementary to the 5’ end of the reverse primer. Mutagenesis...efficiency of assembly decreases as the cloning complexity increases. The efficiency of recombination is...increased with greater homology, so performing more complex procedures can be aided by extending homologous...

...division. These non-plasmid-containing bacteria can compete with and confound the goal of your experiment —...spectinomycin is the more stable and expensive antibiotic compared to streptomycin. Spectinomycin/streptomycin can...option! Otherwise, since it isn’t universally compatible with all bacteria used in research, it isn’t ...express the Tn5 element, but thankfully, most competent cells do not express this factor. Zeocin is handy...host/plasmid DNA during selection if it’s not completely bound up. While this likely occurs at a low rate...Stability Time Saving?  Works for multiple organisms? Compatible with most bacteria? No satellite colonies? ...

...immune system; and reconfiguring of the nuclear compartment, gene expression, and protein translation. Its...producer cells where there is E1 and E3 gene complementation. In target cells that are infected by the vector...virions) are not expressed. Note that replication-competent AdV vectors also exist and are used in certain...topic, which is that natural AdVs, replication-competent viruses with a recombinant genome, and adenoviral...higher than other viral vectors (tens of kilobases; compared to only 4.7 kb for AAV, 9 kb for retroviruses)...Tales: From the Cell Surface to the Nuclear Pore Complex. PLoS Pathog, 11(6), e1004821. https://doi.org/...