...and MEK1 is active or “on.” Dronpa’s color thus indicates MEK1’s current state (Green when off, dark when...photoswitchable Dronpa domains were inserted at sites homologous to the locations they were attached to... a few examples demonstrated in Zhou et al. 1. Testing kinase inhibitors Zhou et al examined the ability...indicator of MEK1 activity. When MEK1 is active, it phosphorylates the ERK KTR and red fluorescence localizes ... fluorescence can be found in the nucleus. For testing kinase inhibitors, psMEK1 and ERK KTR-mRuby2 expressing...blot. Phosphorylation of MEK1/2 peaks at just 5 minutes post-Raf1 inactivation, and then decreases. Treatment...

...autoinflammatory loop: keratinocytes both respond to and release IL-36 cytokines. Keratinocytes are the main source...cytokine family resulting in the activation of keratinocytes, which release inflammatory mediators that trigger...known how IL-36 cytokines actually get out of keratinocytes and induce their effector functions. It’s an...important to study what is happening within the keratinocytes. “We hope to use CRISPR libraries from Addgene...or proteins are involved in IL-36 release in keratinocytes,” says Mellett. This would help the lab identify...

...’s not, I can always take more time to check my notes!  Know your purpose The purpose of our assignment...assignment was clear: present a paper to our classmates so they could understand it. When my mind went blank,...gene of interest incorrectly on slides 8-16. Your notes disappear. The wrong version of your slide deck ...simple as telling yourself, “I can stop and check my notes if I need to,” or “It is okay if it doesn’t go perfectly... field, with very little text on the slides and notes instead of written speeches. When you’re doing all...speak at conferences, or even just provide quick updates during a company meeting increases visibility of...

...a substrate promoting dimerization, Tang et al. tested pairs of their GFP-binding proteins (GBPs) to find...Transcriptional Devices Dependent On GFP). In testing the specificity of their system, Tang et al. found...was absent without electroporated GFP. They then tested T-DDOGs with two mouse GFP reporter lines; again... activation frequency of 56-93%. In the converse test, 98% of TdTomato expressing cells were GFP+, indicating...recombinase, which induces recombination at LoxP sites. Placing a lox-STOP-lox cassette upstream of a gene...T-DDOGs, Cre-DOG is both robust and specific. When tested in retinal electroporation studies, DsRed reporter...

...technical writing and communication skills, Elucidates the big picture and future directions of the ...milestones, possible setbacks, and potential alternative routes, Provides opportunities to convince others why...It typically includes overview information, key dates, funding description, research plan, award information...institution. Moreover, avoidance of ‘to be’ verbs creates strong, well-directed meaning and delivers strength...section. Research challenge The Research Challenge motivates the request for application and offers an opportunity...reaching, remember that you are writing a proposal to test hypotheses and develop novel technologies! If data...

...proteins that cause rare diseases. They now plan to test a large number of other disease-linked Arg-mutant...Engineering the human disease alleles into cells for testing in the lab will be greatly facilitated by CRISPR...Baldwin, Enoch, et al. "Generation of ligand binding sites in T4 lysozyme by deficiency-creating substitutions...function. They will then mutagenize the NFIX gene and test whether variants of unknown significance (VUSs) ...) that are frequently found in genetic testing for syndromes that include developmental delay, result ...dominant negative NFIX phenotypes. Developing a rapid test for the functional activity of VUSs will enable ...

...that blood flows through the heart and lungs and oxygenates the body. However, in neuroscience, this architecture... between neurons are what define how the brain operates, and thus, are a major part of the answer to many...infects one cell (the “starter cell”). It then replicates within that cell, and nascent RABV cross synapses...necessary and sufficient for retrograde transmission (Etessami et al., 2000, Wickersham et al., 2007). Thus, ...answers to the complex questions of how the brain operates. References 1. Callaway EM. Transneuronal Circuit...Mar 19;110(12):E1152-61 Pubmed PMID 23487772. 3. Etessami R, Conzelmann KK, Fadai-Ghotbi B, Natelson B, ...

...which the story demonstrates I possess.   Bullet points on resumes are like cliff notes, so I use these... interviews. I’d also use this notebook to take notes after each interview.   Tailor your 1-hour presentation...rushed and you’ll have to answer questions between bites of food.  Act and look professional  First impressions...and others, if you have their emails. You can use notes you took during your interviews to help craft these...technical difficulties. To try and circumvent this, test out the video conferencing software you’ll be using...

...So, when DNMT1 methylates the genome during DNA replication, it doesn’t methylate sites blocked by dCas9...disease: cancer (Feinberg & Vogelstein 1983). The floodgates, as they say, had opened.  In 2021,  with over...these studies report correlations without clearly testing whether a certain methylation state causes a condition... we need tools to specifically edit methylation sites.  Step aside, transcriptionally active dioxygenase...independent of dCas9 targeting, including modifying other sites in the genome and directly interacting with transcription...

...can mutate the transcription start site, splice sites, exons, and/or promoters to knock out lncRNA function...Bester 2021; Zibitt et al. 2021). Mutating splice sites is particularly effective as it can result in the...Knock-in: Introducing tags or stop signals Once Cas9 generates double-strand breaks, a new DNA sequence can be... be knocked-in, or introduced, into the break sites. Knock-in experiments are important for visualizing...start site (TSS) deletion. (B) Mutations of splice sites. (C) Removal of an exon or a large genomic fragment...RNAs in human cells by Cas9 targeting of splice sites. Nat Biotechnol. Published online November 5, 2018...

...an additional 5–10 minutes of incubation at 37 °C. Wash the membrane for 5–10 minutes 2–3x in fresh wash...solution and agitate for 30–45 minutes at 50 °C. Wash the membrane for 5–10 minutes 2–6x in fresh wash buffer...respectively. Chromogenic detection produces colored precipitates that permanently stain the membrane, making ...membrane in stripping solution and agitate for 5–30 minutes at room temperature. The incubation time will depend...

...cleavage at 1-12 off-target sites; in contrast, SpCas9 may cleave at ~90 sites, according to Kim et al. ...in AT-rich genomes. Additionally, whereas Cas9 generates blunt ends 3 nt upstream of the PAM site, Cpf1... strength may lie in its low off-target editing rates, determined using sophisticated genome-wide analysis...many of its computationally predicted off-target sites, Cpf1 does not mediate detectable off-target cleavage...mechanism. To prevent new editing post-HDR, repair templates should remove the PAM sequence. Many research ...

...recommend no more than 40 minutes for the panel discussion leaving 20 minutes for audience questions (30...Follow Addgene on LinkedIn for repository news and updates. Panel discussions are becoming a ubiquitous format...members all say the same thing and one panel member dominates the discussion? Or maybe the one that goes way...interviews, the moderator should be taking good notes and discovering interesting stories, riffs or opinions...be good at talking, so they will easily take 5 minutes to introduce themselves and, voila, 1/2 of your...

...recombination using a repair template. These templates can include loxP sites to create conditional floxed alleles... targeting vector such that a set of three LoxP sites flank the resistance gene and the targeted exon ...replaced with the same sequence flanked by LoxP sites while the resistance cassette has been placed into...marker using Cre recombinase. Since there are 3 loxP sites, recombination can occur in multiple ways. The desired..., as seen in line 4 of Figure 4. Since the loxP sites are located in intronic regions, this gene will ...

...DNA into smaller pieces, and proximity ligation creates chimeric circles of DNA that are distant linearly...delivery of the two remaining CAPTURE 2.0 components eliminates the need to create stable cell lines and allows... beta globin locus. The two methods had similar rates of specificity for the DNA sequence targeted in ...interactions at once. Chromatin interactions at five sites in the well-characterized beta-globin locus were...negative control gRNA or predicted gRNA off-target sites. Long-range chromatin interactions identified with... a more vivid chromatin picture, CAPTURE 2.0 re-writes the script on gene regulation. References Han...

...serotype AAV1. Distinguish dendritic neurites from axonal neurites with AAV vectors expressing cytoplasmic... the antibody toolbox, they screened antibody candidates using a synthetic human scFV library and characterized.... After finding no cross-reactivity, antibody candidates underwent affinity screening to select for slow...slow off-rate binders. The best candidates were validated by IP-MS using HEK293 cell lysate where the ...providing enzyme protection while still allowing substrates and products to diffuse freely into or out of...

...intracellular trafficking, and protein turnover rates. Additionally, by engineering FP fusions associated...individually generate gRNAs and homology arm repair templates containing the tag for each insertion. Also, it...digestion of the destination vector with SapI yields 2 sites- the first site accepts the sgRNA target sequence...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... vectors for inserting a tag at multiple target sites. SapTrap vectors can be easily modified to tag hundreds...

...to Microbial Media BitesizeBio: How to Get Better Plasmid Midiprep Yields BitesizeBio: Better Plasmid Midipreps...figure at right for an example). Look out for carbohydrates Certain strains, including HB101 and derivatives...derivatives, release large amounts of carbohydrates that can inhibit the cell lysis process. To ensure that...may lead to loss of the plasmid, and using older plates could increase plasmid loss or mutation. Let it...smaller cultures, especially those in deep-well plates, require a faster shaking speed for proper aeration...

...an antigen binding site for a total of 2 binding sites per antibody. C) IgG’s are big enough that multiple...a double-edged sword. An antibody’s large size creates more distance between the target antigen and the...process can be avoided direct antibody fluorophore conjugates are used, but these are usually more expensive...varieties. This refers to the number of cysteine sites the nanobody has available for conjugation to a ...commercially available. References 1. Pleiner, T., Bates, M., & Görlich, D. (2017). A toolbox of anti–mouse... plasmids from this publication at Addgene. 2. Bates, M., Böhning, M.I., Chug, H., Görlich, D., Lee, ...

...construct (i.e.,functional cassettes are recyclable) Can only work if the restriction sites used for the assembly...repeatability of the method. Note that the restriction sites of the MCS are only regenerated at the 5’ and 3’...free of specific restriction enzyme recognition sites. Thus, chaining methods are not necessarily suitable...sequences. Intermediate chaining products (called “cassettes”) can be readily reused in the assembly of any... et al., 1962), SalI, XhoI and MluI recognition sites, which contain CpG dinucleotides in their 6-mer ...