... and, if you do it right, can be (part of) figure 1 of your next paper or thesis. This series of posts...An explanation of the assay is depicted in figure 1. The strict correlation between the fluorescent protein...brightness When a dish of mammalian cells is transiently transfected with a plasmid containing a fluorescent...fluorescent proteins, e.g. see Chudakov et al (2010), Cranfill et al (2016) or Thorn (2017). However, the theoretical...developed in our lab uses a reference protein that is translated from the same open-reading frame and separated...reference protein) under identical conditions, a ranking of the practical brightness can be made (Goedhart...repeating this with another fluorescent protein, a ranking of practical brightness can be made. This strategy...

...modified slightly with either a 1-7 base poly(U)tail in tRNA-gRNA constructs or a 1-4 base tail from the second...tRNAs contain internal transcriptional elements that enhance pol III transcription, potentially increasing...base Csy4 recognition sites from a polycistronic transcript. Although this method shortens construct size...coexpression of Csy4 with Cas9 and the polycistronic transcript. As nucleases may have off-target effects and...matching traditional gRNA-expressing constructs, transcript levels of PTG1 and PTG2 were 3 and 31 times higher... of polycistronic gene organization, and its translation to CRISPR/Cas9 shows great potential for multiplexed...

... to focus, which was extremely effective. Others ran experiments at odd hours, used more secluded workspaces...consistently and accurately run a protocol.   Figure 1: Evolution of a Western blot protocol, from a full...

...non-functional allele. For a 1 kilobase gene, since potential target sites occur ~1 in every 8 nucleotides,...10.1038/s41576-018-0059-1 Veres A, Gosis BS, Ding Q, Collins R, Ragavendran A, Brand H, Erdin S, Cowan CA... efficacious to target the coding or non-coding strand of DNA. As a rule of thumb, we avoid target sites..., 2020). is not limited to single nucleotide transitions but still requires a nearby PAM, although these... modulating gene expression at the level of transcription – CRISPRa (activation) and CRISPRi (inhibition...most-efficacious to target a ~100nt window upstream of the transcription start site (TSS), while for CRISPRi, a ~100nt...of producing the guide (synthetic, in vitro transcription, or lentiviral delivery) can affect the accuracy...

...are working with is correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...the specific reaction you are running. Transformation Transform a few microliters of your ligation reaction...The PCR could fail to produce a product, the transformation may not result in any cells, or all the colonies...fragments If you don’t get any positive clone, try transforming the ligation mix in different bacterial strains...

...method entails arranging all calculated p values from smallest to largest and ranking them (1 being the smallest...error, variance, data ranges. how closely data points fall to each other (see Fig. 1). Alpha values –...statistical test assumes your data follows. Fig. 1: Examples of comparable groups with and without equal...significance by finding the largest p value where its rank divided by the total number of comparisons performed...– Tukey’s test, Newman-Keuls, Duncan’s multiple range test, and several others. Each test has different...negatives, moderate false positives. Duncan’s multiple range test – Most powerful. Low false negatives, high ...): 126-130 (2012). 10.1097/SMJ.0b013e3182498ad5 Curran-Everett, Douglas. Explorations in statistics: statistical...

...to reach out to potential depositors in two ways: 1. Fill out our "Suggest a Plasmid" form Fill out the...where we can find the plasmid. While we cannot guarantee that everyone we reach out to will end up depositing...

...protein of interest will be problem-free. In figure 1 we provide an example of the OSER assay with a number...can be shared by publishing it as (part of) figure 1, uploading it as a preprint, or documenting it in ...not by Cranfill et al (2016)). Moreover, good performance in the OSER assay is not a guarantee that using...fluorescent proteins when protein tagging is the goal (Cranfill et al, 2016; Constantini et al, 2012). The localization...

...immunogenicity score (Mattei et.al., 2022).    Figure 1: Generation of immunogenicity scores by predictive...antibodies for the treatment of diseases. J Biomed Sci 27, 1 (2020). https://doi.org/10.1186/s12929-019-0592-z  ...ADAs can inactivate the drug and increase drug clearance, lowering its efficacy and increasing the risk...

...Glycine-HCl 25–200 mM SDS 0.1%–1% (w/v) Tween 20 (optional) 1% (v/v) HCl Adjust to pH 2.0... your gel and membrane transfer as normal. Immediately after transfer, dry the membrane to maximize protein...you from having to run a whole new gel and membrane transfer. Let’s get started! Before you begin In the...manufacturer’s instructions for the membrane you are using. Dry nitrocellulose membranes are brittle, so proceed ... as normal! Stripping your membrane Alright, so you’ve dried your membrane and returned it to buffer, ...’ll want to: Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...a fume hood. Rinse the membrane in water or fresh buffer. Cover the membrane in stripping solution and...

... of two outcomes (see schematic depiction below): 1. Resistant cells among the separate replicates would...development of combination therapies.         References 1. Hyo-eun, C. Bhang, et al. "Studying clonal dynamics...library was developed when Carrie was a post-doc in Frank Stegmeier’s lab in Novartis Oncology.  Bhang did...lentiviral vectors with roughly 73 million semi-random, 30 bp DNA barcodes that are integrated into target...

...none. Best for: targeting single stranded RNA.   Table 1: Fast facts on Cas family proteins...The main differences between Cas7-11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity.... it will proceed to generate a blunt DNA double strand break. Cas9 has been studied for genome engineering...an expanding number of others. Best for: a wide range of DNA edits.   Cas3 – The shredder What sets Cas3...locates its target sequence and initiates single stranded DNA degradation. Cas3 DNA digestion can proceed...convenient for targeting experiments requiring single strand DNA generation, such as homology directed-repair...therapeutic delivery.   Cas14 – Mini, but single strand mighty Unlike many other Cas enzymes, Cas14 targets...

... Hot plasmids covered in this segment Hot Plasmid 1: B. subtilis knock out and knock down libraries. Listen...on the B. subtilis libraries. Hot Plasmid 2: Transcription factor fusions modulate iPSC reprogramming efficiency...

...  Hot plasmids covered in this segment Hot Plasmid 1: Photocleaveable PhoCl protein for controlling molecular...Hot Plasmid 3: CRISPR base editors with enhanced range and specificity. Listen to to the Segmennt of Improved...

...Hot Plasmid 1: The Tol2 Gateway-Compatible Toolbox for quickly generating zebrafish transgenic lines to ...

...874 . https://doi.org/10.1038/nmeth.3993  Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu.../doi.org/10.1038/s41586-019-0908-x  Nishimasu H, Ran FA, Hsu PD, Konermann S, Shehata SI, Dohmae N, Ishitani...949. https://doi.org/10.1016/j.cell.2014.02.001  Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz...indel formation and phenotypic changes as early as 1 week post-injection. Livers from these mice were histologically...gene delivery due to their low immunogenicity and range of serotypes allowing preferential infection of ...prime editors. While using AAV to deliver large transgenes might seem daunting, it’s actually a challenge...split AAVs. In general split AAVs break a large transgene in two pieces and package each piece into an individual...

...all of its successes, has many deep-rooted flaws (1). As we discovered through the four workshops at the... Follow David on Twitter @driglar.     References 1. Alberts, Bruce, et al. "Rescuing US biomedical research...Ambika, et al. "Transforming training to reflect the workforce." Science Translational Medicine (2015)...better solution would be to reduce the number of entrants into PhD programs to more closely reflect the ...symposium. Eve Marder, professor of Biology at Brandeis University, has recently published an article ...Among other involvements, he helped to establish grants through the Sloan foundation for postdoc-related...

...2021.   The AID system relies on a transport inhibitor response 1 (TIR1) expressed under a desired promoter...and accurately in a “single pot.’   Figure 1: Single-Pot Assembly: a) Graphic depiction of chromatin...editing assemblies are sensor-actuators, either transcriptional regulators or chromatin-modifying enzymes. ...cell surface. When the cells interact, GFP is transferred to the receiver cell, and the receiver cell turns...These libraries encode target sites flanked by randomized nucleotides in place of a protospacer adjacent.... Check out the new additions.  Image calcium transients in PV interneurons with AAV-S5E2-GCaMP6f, now...

...scavenger hunt and the subsequent clue. (Begin with clue 1 below!) You will find five photos this way. At the...Subscribe to the blog to stay in the know!     No pranks, just fun and games this April Fool’s Day on the... to find pictures of Addgene pets with Addgene-branded clothing on the blog. Below each photo, the pet...

...company culture.  It’s also a good idea to prepare 1-2 unique questions for each interviewer by looking...to take notes after each interview.   Tailor your 1-hour presentation to your audience Start by asking...position, I just had a phone interview. I also transitioned between different lab groups without having ... an opportunity to demonstrate technical and transferable skills that make you qualified for the position...experience less stressful. First, you can make arrangements with those you live with to have a quiet place...