...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...

...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...

... 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 ...

...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...

...some data, etc.  The firestorm that followed implementation of the policy comprised of numerous objections...raw data requires huge amounts of storage. For example, mouse behavioral geneticists may track the motion...although it might be argued various authors have simply chosen to submit elsewhere). The majority of the...numbers of figures such that many have multiple, complex panels. PLOS, itself, has not finalized its definition... paper are in scope. For images, the number of examples supporting the published material is a factor ...types? And how long should data be archived? Simplifying data access Some of the above mentioned issues...collections and interpretation of data, publishing complete data, rather than just a single projection of ...

... relatively simple, changed the course of scientific research. What was once a complicated process of ...and requesting scientists, all of the Addgene employees, the Addgene board members, vendors, Addgene supporters...prototype of an interactive game that Addgene employees created and designed for the Hall of Human Life...playing all night was hand-picked (like our plasmid samples!) by Addgenies. If you were there, you might have... on sliders, hot dogs and a mashed potato bar (complete with all the fixings), as well as plenty of delicious...of requesting and sharing plasmids has become simplified through Addgene. Looking back, Addgene’s journey...

...to the popularity of GFP including (i) fast and complete maturation to functional, fluorescent protein ...endogenous fluorescent co-factors can be used. Examples include the LOV domains that bind flavin (Buckley... pKa, which is around 6.0 for GFP. This number implies that at pH 6 only 50% of the available green fluorescent...under acidic conditions, with pKa values below 4. Examples include mTurquoise2 (pKa = 3.1), tagRFP (pKa =...diameter of about 2.4 nm (Hink et al., 2000). The complete beta-barrel is necessary for its fluorescence ...specifically and with high affinity to fluorophores. Examples are the FlAsH tag, the His tag and several others... proteins are also a better choice for thicker samples. Another solution to avoid autofluorescence is ...

...then following transduction, the partial genomes complement each other to restore the full-expression cassette...strategy for increasing the size of the AAV donor template for CRISPR-mediated homology directed repairs ...co-transduced with multiple copies of both vectors. Examples of use: Halbert et al., 2002 used an overlapping...outperform single oversized AAVs (Hirsch et al, 2013). Examples of use: Lai et al., 2005 used a trans-splicing... the accumulation of such truncated proteins. Examples of use:  Ghosh et al., 2008 used a hybrid split...homologous recombination approach delivers large donor templates using two sequential homology directed repairs... the gRNA target site that mediated the donor template’s integration so that the site is reconstituted...

...Palindromic Repeats. That contrast of simplicity and complexity is reflected in the biology, too. CRISPR..., and the spacer sequence is used to target complementary DNA sequences. In laboratory settings, an sgRNA...whose sequence is not checked. Assuming full complementarity, its sequence matches the guide sequence. PAM...into a single strand that folds back on itself, simplifying the CRISPR-Cas9 system for laboratory applications...either crRNA or sgRNA). Cas9 requires perfect complementarity in this region between the guide and target...post on the structural biology of Cas9. Want a simpler guide to keep at your desk? We’ve got a CRISPR ...

...GPCRs (Takeda S et al., 2002) and many downstream complexes involved, it can be difficult for scientists to...in sight (Olsen RHJ et al., 2020). A transducer complex consists of a trimer of three subunits: alpha, ...collectively known as a heterotrimeric G protein. This complex converts a signal received by a GPCR on the cell...is being able to identify specific transducer complexes that are activated in response to a ligand binding...inactive, these subunits are found in a trimeric complex bound to the intracellular surface of the GPCR....Following activation in response to the ligand, the complex will disassociate, resulting in a decreased GFP2...screening across the transducerome, to gain a more complete understanding of a GPCR mediated response.  Acknowledgements...

...communicating science to laypeople.  Laypeople are simply non-experts in the field of science you are communicating...use-case (definition) in a specific field.  For example, while “PCR test” has recently transitioned from...PCR test” to mean “a test that uses a biological sample to look for presence of a virus.” A scientist would...sequence that uses trprimers and enzymes that will amplify a sequence only if present.” You can see how you...the possibilities are endless.    Activity Example audience  Talking about your career as a scientist... podcast about genetics using dog breeds as an example adults 45-55 yrs old who breed or are interested...online patient/caregiver communities  Table 1: Examples of defining your audience for science communication...

...will result in more engaged, successful employees who can accomplish great things.   To read more on this...time for that and you won’t be as productive. For example, the many resources available on the Addgene website...enough to do…you are not delegating enough. For example, if we need to develop a new plasmid collection...duplicated. With each component of the project completed by an individual who is a local expert in that...result is high quality and the entire process is completed more efficiently. Listen to our podcast interview...desired outcomes. Concern yourself with what is accomplished, rather than detailing how the work should be...