... https://doi.org/10.1038/s41556-021-00836-1 Visual barcodes for live cell clonal multiplexing by: Rachel... Zebrafish) (Xiong, et al., 2021).    Fig. 1: Schematic showing the workflow for the BLITZ system... error rate for long sequence knock-ins Visual barcodes for live cell clonal multiplexing Improving ...nanobody-GFP interaction). The modularity of the system provides researchers better flexibility and makes the approach...other experiments. And, while the BLITZ system was designed and optimized with zebrafish in mind, it can easily...Recently, Ravid Straussman’s lab has developed visual barcodes for clonal multiplexing of cells in live imaging...peptide. By combining three different localization peptides with four different fluorescent proteins, and ...

...Introduction to Biosensors. Essays Biochem. 2016 Jun 30;60(1):1-8. DOI: 10.1042/EBC20150001 Leopold, A. V., Shcherbakova...have pre-designed plasmids for these systems! Ion sensors One common biosensor class is designed to monitor...all have specific sensors designed for them. For example, this class includes tension monitors associated...interest for many disciplines! Most of the sensors designed for calcium generate a fluorescent output, which... fluorescence in the presence of calcium. This includes FRET-based sensors such as Twitch. While calcium.... Physiological factors Unlike the previously described sensors, this class measures either factors as...to use them, you’ll need to use the vectors (or redesign the tools). It’s a good thing, though! Viral delivery...

...correct. It's also best to use 1-2 ug of the vector for digestion. PCR Design primers that overlap at least...plasmid; a starting point could be a molar ratio of 1:2 (plasmid:insert), but this parameter has to be optimized...coveted plasmid with no substantial delays. Design, design, design Before diving into the experimental work...experimental work. Fragments generation After you’ve designed your cloning scheme, you’re now ready to generate...from a large genome, you can recheck your primer design or optimize your primers or PCR conditions. Purifying...second and more detailed look at your experimental design. Full lawn of cells. The antibiotic in your plate...complementary to standard sequencing primers. If not, design primers to sequence your insert. When you get your...

...central ɑ-helix that can be split into 3 parts: (1) β-strands 1-9 (β1-9) and the one ɑ-helix, (2) β-strand ...fluoresce: FlipCherry. FlipCherry provides a proof of concept that the designed used in FlipGFP can be applied...range and high brightness. The Shu Lab from UCSF designed an improved apoptosis reporter, ZipGFP, which ...prevent binding, the β strands in FlipGFP were redesigned so that they are parallel to each other and thus...fluorogenic reporter toolkit, the Shu Lab also designed a red fluorogenic protease reporter based on superfolder...PMCID: PMC2117903. To, Tsz-Leung, et al. "Rational design of a GFP-based fluorogenic caspase reporter for...Central PMCID: PMC5026494. Zhang, Qiang, et al. "Designing a green fluorogenic protease reporter by flipping...

...'ll want to design a custom primer. Because Sanger sequencing can usually sequence just 1 kb of DNA, custom...screening blog post for more information.   Figure 1: Result of a blue-white screen. Image from Stefan ...is not enough on its own to help you find your desired plasmid. In many cases, you will need a screening...you will need to sort through them to find the desired clone. Do your selection, then screen Why add a...what about identifying clones that contain your desired insert? This is where a  screen comes in. Let’s...blue-white screen, which relies on the lacZ gene. lacZ encodes the enzyme 𝛽-galactosidase which can hydrolyse...Sanger sequencing determines the precise order of nucleotides within a DNA molecule, in this case a plasmid...

...additional flexibility in pseudotyping.   Figure 1: Genome of a prototypical gamma-retrovirus.    ...an SV40 promoter, and Neomycin selection.   Table 1: Gamma-retrovirus glossary Plasmid Type Element...infectivity): The use of the VSV-G envelope provides the widest tropism or range of cells a virus can infect...The widespread use of γ-retroviruses (gamma-retroviruses) in cancer and stem cell research has prompted...lentiviral packaging methods. The three components described above (envelope, packaging, and transfer) are ...into the 293T packaging cell line. This system provides the greatest flexibility to pseudotype γ-retrovirus...nomenclature of the plasmid backbone is meant to be descriptive of the components found within the backbone. ...

...versions with toxicity to many organisms. Fig. 1: Exogenous sources of dsRNA Cellular sources of... 286-301 (2022). doi.org/10.1038/s41580-021-00430-1 More resources on the Addgene blog CRISPR 101: RNA...into agriculture as species-specific pesticides and insecticides, an advancement over previous chemical...prone to degradation – those are the words that describe RNA! Double stranded? Well, that’s just for DNA...also known as RNAi, are short, dsRNA molecules designed to degrade mRNA transcripts. RNAi activates the...sequence is found (typically RNA that the RNAi is designed against), RISC can repress translation and/or ... and repetitive sequence. All of the R-loops described above play distinct roles in promoting cellular...

...This project: 1) develops state-of-the-art methods of sex and gender analysis and 2) provides case studies...healthcare system was $35.2 billion (or $140 per $1 invested in the trial). The goal of gendered innovations...approaches to gender equality over the past several decades: “Fix the Numbers of Women” focuses on increasing...using both sexes in basic research is excellent design), but they used all female stem cells—this was ...environmental factors. How to include sex in experimental design Recognizing the importance of analyzing sex, the... above, another excellent resource for research design is Kalpit Shah et al., “Do you know the sex of ...Corporations need to integrate these insights into product design. But eyes have been opened—and we cannot return...

... 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...monitored over time by sequencing the barcodes in the population (all barcodes can be amplified using the same...with roughly 73 million semi-random, 30 bp DNA barcodes that are integrated into target cells' genomes...ClonTracer system, lentiviruses containing these barcodes infect the target cell population at low multiplicity...replicate populations, they found that similar barcodes consistently made up the majority of the resistant...resistant populations. In fact, 88% of the barcodes within the resistant populations were shared by at least...

...addition of nucleotides to the 5’ spacers. The 3’ ends were modified slightly with either a 1-7 base poly...poly(U)tail in tRNA-gRNA constructs or a 1-4 base tail from the second tRNA in the tRNA-gRNA-tRNA constructs...advantage over other methods that favor certain nucleotides at the 5’ end. PTG construct schematics. Scissors...the plasmids made by Xie et. al are specifically designed for plant expression, PTGs will likely be adaptable...worries, I've summarized the three strategies described in this post in the graphic below - including ...

...plasmid, based on the assumptions outlined in Table 1.   Of course, this is for the simple insertion of...any restriction enzymes or vector purchases (Table 1). Restriction enzymes and vector purchases can raise...in science – the time spent by the scientists designing and performing the experiments. This cost varies...quickly with the postdoc who’s really good at primer design – but first, you have to know what resources exist...well) plate! How much do repositories help?   Designing precise calculations for any of the numbers in...engagement with and use of repositories like Addgene provides large-scale savings. How large? Using Doench’s...

...introduced, they can deplete endogenous protein levels for 1-3 days. There’s also a CRISPR for that! CRISPR interference...more about them on our CRISPRa blog! Fig. 1 CRISPR activator and repressor mechanisms Want...and SMASh degron tagging vector! Destabilizing domains Destabilizing domains (DD) are akin to degron ...   Fig. 2 Tetracycline on/off promoters and modes of action Check out Addgene's Lentiviral Tet-on ...post-translational level, unlike most of the other mechanisms described. Fig. 3 dTag mechanism of protein degradation...

... closely data points fall to each other (see Fig. 1). Alpha values – numerical risk allowance in a ...statistical test assumes your data follows. Fig. 1: Examples of comparable groups with and without equal...values from smallest to largest and ranking them (1 being the smallest value). It calculates the cut off... been described in this blog. If your data does not meet the assumptions for the tests described above...experimental groups, but how do you determine if they deserve that oh-so coveted asterisk of significance? You...referred to as two-sample or independent, but these designations all refer to the same test. On some occasions...Correction and the Benjamini-Hochberg procedure are designed to reduce false positive rates and control for...

... 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...tables with fluorescent protein properties and provides a quick way to compare fluorescent proteins, e.g...been used in yeast by Lee et al (2013) and in nematodes by El Mouridi et al (2017) and Heppert et al (...practical brightness. Therefore, practical brightness provides a better picture of what can be expected in a ...

...Last updated Sept 1, 2020. This post was contributed by David Wyatt and Dale Ramsden, UNC at Chapel Hill...the course of repair by NHEJ are typically small (1-10 bp) but extremely heterogeneous. There is consequently...and does not consider the missing or damaged nucleotides that are common to biological sources of DSBs...better substrates for ligation. Although we do not describe these steps here, the processing of DNA ends tends...of the canonical Cas9 approach. A pair of CRISPR guides that flank regions of hundreds of base pairs or...

...of an industry H-1B need to be aware of the April 1 deadline for companies to submit an application on...and non-profit organizations do not have the April 1 H-1B queue process or quota like industry, these organizations... National Interest Waiver. The three prongs are: (1) The foreign national’s proposed work endeavor must...-1B, you need to prove 3 threshold requirements: (1) that you have international recognition for your ...The recently established case Matter of Dhanasar provides the three-prong test that US Immigration officers.... As Immigration is a federal law, Casseus Law provides legal services to people located in all 50 states...

...overhangs may be present in the original plasmid (Option 1) or added using PCR-based amplification (Option 2)...Methods Mol Biol. 2014;1116:119-31. doi: 10.1007/978-1-62703-764-8_9. PubMed. More Resources at Addgene:  ...customized to direct assembly of DNA fragments. When designed correctly, the recognition sites do not appear... scheme is as follows: the gene of interest is designed with Type IIS sites (such as BsaI or BbsI), that...and do not appear in the final construct. The destination vector contains sites with complementary overhangs...ligation can be done in one 30-minute reaction. The destination vector and entry vector(s) are placed in a single...IIS enzyme and ligase. Although the original destination vector + insert may spontaneously religate, this...

...several advantages over traditional vaccines, including 1) cellular responses in addition to antibody response...and immunogenicity of attenuated, nef-deleted HIV-1 strains in vivo. Retrovirology. 2007 Sep 23;4:66. ...biology – they can deliver materials to specific destinations, haul a variety of cargo, and even handle a ...delivery of the actual virus to which immunity is desired. Instead, components of the virus are now delivered...

...effective with as little as 3 ng of DNA!) Figure 1: Schematic of SLIC. To start the SLIC cloning process...Methods Mol Biol. 2012;852:51-9. doi: 10.1007/978-1-61779-564-0_5. PubMed. JBEI-J5’s guide to SLIC Resources..., the use of LIC is often limited to specially designed plasmids. Sequence- and ligation-independent cloning...the “damage” and producing a complete plasmid. Besides being compatible with any vector, the SLIC protocol...

...part of) figure 1, uploading it as a preprint, or documenting it in some other way. Besides the data, new...protein of interest will be problem-free. In figure 1 we provide an example of the OSER assay with a number...Amsterdam. The previous two posts in this series described a practical approach to selecting a bright fluorescent...