...of interest is relatively small, adding GFP may alter diffusion rates. Smaller genetically encoded probes...cofactor binding proteins as described above. The smallest tags are peptides that are engineered to bind ... For a review see Lotze et al. (2016). To date, small probes that are entirely genetically encoded do ...in the medium or flavin containing proteins in mammalian cells. Generally, autofluorescence is less of .... "A genetically encoded fluorescent probe in mammalian cells." Journal of the American Chemical Society...bioluminescence imaging of deep tissue in freely moving animals." Science 359.6378 (2018): 935-93. PubMed PMID:...

...’ll want to run gradients for optimal annealing temperature and optimal primer concentration; check for...parameters, some people find it can detect even smaller differences, and it can even be used to detect ...chemistries available and they can be the more optimal choices at times. For example, chemistries that... plate type or tubes, sealing method, and other small variations in consumables. Track and record these... passive reference dye, such as ROX, can help normalize for that. Passive reference dyes are inert fluorescent...machine surge errors and allow for well-to-well normalization of signal. They are often included in commercial... a protocol that automatically performs the normalization step.  Machines Most general-use protocols, ...

...to occur at a frequency of 1.2 per megabase in mammals), leading to DNA damage and developmental aberrations...indicating that the recombinases were inherited normally and thus nontoxic. In contrast, 0/79 offspring...two of these recombinases were also active in mammalian cells. In experiments, B3 and KD were shown to...site-specific recombinases for use in manipulating animal genomes. Nern A, Pfeiffer BD, Svoboda K, Rubin ... and DNA damage induced by Cre recombinase in mammalian cells. Loonstra A, Voojis M, Beverloo HB, Al Allak.../s004270100167. PubMed. Cryptic loxP sites in mammalian genomes: genome-wide distribution and relevance...

...targeted region, as shown below.  To identify optimal nickase designs, IDT scientists Mollie Schubert...consistently higher than that of H840A, especially with smaller nick distances. Using D10A, they subsequently showed...the PAM-out configuration, D10A tends to generate small deletions, while H840A is biased towards large insertions...and Yan next examined a situation thought to be suboptimal for HDR. At the AAVS1 locus, the two nearest ...13 nt away from the target site rather than the optimal <10 nt distance. Employing two gRNA sites that ...between nick sites Use 40 nt homology arms for small insertions or tags (ssODN; IDT Ultramer® Oligonucleotides...

...functions in mammalian expression systems.   Christopher Newgard’s Lab has fused LSD1 to the smaller nuclease-dead...specified loci. Charles Gersbach’s lab has deposited mammalian expression constructs including pcDNA-dCas9-p300...Tet1-CD for targeted cytosine demethylation in mammalian cells. This plasmid is used with pcDNA3.1-MS2-...DNMT3A-PuroR for targeted cytosine methylation in mammalian cells. Co-expression markers EGFP and PuroR enable...pcDNA3.1-dCas9-MQ1(Q147L)-EGFP, a fusion of dCas9 to a small DNA methyltransferase from the prokaryote Mollicutes...Jaenisch R (2016) Editing DNA Methylation in the Mammalian Genome. Cell 167:233–247.e17 . https://doi.org...

...i-GONAD is performed directly on pregnant female mice using a small incision that exposes the ovaries and ...into blastocysts to be implanted in pseudopregnant female mice. If you obtained the desired chimeric mice...template with 55-100 bp homology on each side. For optimal repair, place the loxP sites precisely at the cut...microinjection and i-GONAD knock-in efficiency using a small ssDNA, and they found comparable efficiencies (52...However, microinjection does require 2.5x more animals than i-GONAD and is a more technically difficult...for Cas9 Purification Find CRISPR Plasmids for Mammalian Expression ...

...calculated p values from smallest to largest and ranking them (1 being the smallest value). It calculates...test. Distribution - spread of data; typically, normal (bell curve) or skewed (see Fig. 2). Assumptions...   Parametric test assumptions: Distribution – Normal Variables – Independent Variance – Approximately... the assumptions for the tests described above (normal distribution, equal variance, and independent variables...histogram is sometimes sufficient to determine normality (or lack thereof). Alternatively, a Quantile vs...from a theoretical distribution. If your data is normally distributed, it will form (more or less) a straight...

...expression vectors are available at Addgene. A set of mammalian expression vectors containing various spike protein...nonstructural proteins 1 to 16, spike proteins, envelope small proteins, and membrane proteins. Although all viral...modification may not be easily expressed in non-mammalian cells. Yeast expression plasmids The yeast expression...cleavage, codon usage, and expression system. Mammalian expression plasmids Finally a set of wild type...transforms, innoculates, and performs DNA minipreps for small batches of samples. As soon as that process is complete...data! These same plasmids are then added into our normal full plasmid NGS queue in order to bring them inline...

.... To overcome this, most scientists use mutant animals that are genetically altered to overexpress endogenous...labeling (PL) uses a promiscuous labeling enzyme and a small-molecule substrate to map the spatiotemporal distribution...PL were APEX2 and BioID. APEX2 is fast, tagging proximal proteins within minutes, but requires H2O2, which...temperature range than BioID, which only functions optimally at 37℃. The increased efficiency of TurboID and...easily concentrated. Viral RNA is then amplified isothermally, and detected through a toehold switch, an RNA...the purified domains include protein stability, small molecule binding screens, peptide arrays, and phage...

...method, we recommend basing your selection on a small-scale head-to-head comparative study under conditions...calculated by multiplying EC by QY (EC*QY, sometimes normalized to the value of EGFP). The higher the number,...Determining practical brightness When a dish of mammalian cells is transiently transfected with a plasmid...in the fluorescence intensities of individual mammalian cells within the dish. This is caused by the stochastic...Another way to dodge cellular variation observed in mammalian cells is to tag an endogenous gene. By imaging...Mouridi et al (2017) and Heppert et al (2016). In mammalian cells this would be possible with CRISPR/Cas-based...

...This post, along with a future companion post on mammalian vectors, will catch you up on the core replication...cell structures similar to those of plants and animals. Unlike bacteria, yeast can post-translationally...CEN). These vectors replicate as though they are small independent chromosomes and are thus typically found... vectors are stable without integration. Yeast Episomal plasmids (YEp): These are most similar to bacterial... by the plasmid may be expressed at higher than normal physiological levels due to high copy numbers. ...and ability to be used in bacterial, yeast, and mammalian cells.   ...

...the full genome CRISPR screen approach is to use smaller libraries targeting subsets of genes. With such...where CRISPR modified cells are transferred into an animal and the behavior of the transferred cells is followed...of candidate genes from a full genome screen, a smaller, secondary, CRISPR screen could be used to validate...Addgene, and then use Green Listed to design a smaller validation screen using selected gRNAs extracted..., studying genes affecting cellular behavior in animal model systems. Use Green Listed to extract gRNAs...), Yusa (5), Wang/Lander/Sabatini (6), and Chari/Mali/Church (7). These reference libraries contain multiple...much, showing that we are far from consensus on optimal gRNA design. With Green Listed you can also easily...

...to the force to control protein expression in mammalian cells: easy to use biological tools are abundant...when knockouts aren’t viable). siRNA or RNAi are small, double stranded RNA molecules that degrade target...by placing tet response elements upstream of a minimal promoter. These systems come in two varieties: ...degradation. Since the tagged protein of interest is normally expressed, protein levels will quickly recover...the ligand is removed. Degron tag systems include small molecule assisted shut off (SMASh), auxin-inducible... and quickly degraded. However, if the relevant small molecule is introduced into the system, it will ...

... Their production requires animal sacrifice and because they are an animal derived product, there can ...limitation and allows for the flexible design and small scale production of antibodies. Problems with antibodies...Genetic Tagging - Since antibodies are produced in animals instead of via recombinant DNA, they can’t be genetically...and better penetration of the sample due to the smaller size of RANbodies compared to antibodies. Staining...Reporter-nanobody fusions (RANbodies) as versatile, small, sensitive immunohistochemical reagents. PNAS. PubMed...

...infected a non-small cell lung cancer cell line containing an activating mutation in epidermal growth factor...researchers to individually label millions of mammalian cells through lentiviral infection and to monitor... other replicate; prior to treatment, there are small (previously undetectable) populations of cancer ...combination treatment with multiple drugs reveals even smaller preexisting populations with resistance to both...distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition." Nature medicine...

...last updated Jul 12, 2021. What started out as a small collection of plasmids for coronavirus research,...They developed a method that uses loop-mediated isothermal amplification after reverse transcription (RT-LAMP... reaction to detect SARS-CoV-2 nucleic acids. Mammalian targets During the 2003 SARS outbreak, Hyeryun...the virus interacts with human cells. Addgene’s mammalian target plasmids express these and other targets...many expression systems: bacterial, yeast, and mammalian. These plasmids contain both untagged and tagged...2021.04.22.440848 Crawford KHD, Eguia R, Dingens AS, Loes AN, Malone KD, Wolf CR, Chu HY, Tortorici MA, Veesler D, Murphy...

...create a double-strand break works well because very small deletions and insertions in the DNA sequence can...nucleotides in length and lack an open reading frame, so small deletions or insertions may not result in a functional...degrade targeted lncRNAs. The Cas14 variant is a smaller and more versatile option. It can be used to target...specificity, particularly in applications requiring smaller delivery systems (Zibitt et al. 2021). Even though... Cas13 Targeting RNA with Cas13a (C2c2) Cas13d: Small RNA-targeting CRISPR enzymes for transcriptome engineering...10.1038/nature20149 Jiang Q, Meng X, Meng L, et al. Small indels induced by CRISPR/Cas9 in the 5’ region of...

...interaction between the fluorophores and serves as a small-scale ruler to measure distances that are too miniscule...orientation of the donor and acceptor proteins is not optimal, FRET may not occur or be detected even if the ...experimental setup as a potential source of error in suboptimal or undetectable FRET, well-characterized FRET...need. FRET biosensors designed to measure specific small biomolecules or gene activity are often intramolecular...interactions, measure the concentration or activity of small molecules, detect cellular processes and signaling... lead to monitoring of these processes in live animals (Hirata and Kiyokawa 2016), and molecular tension...

...are read after each cycle. The raw data is then normalized to a reference sequence present in the same amount...housekeeping’ gene), and quantified relative to a 'normal' sample with known copy number set as the expression... all qPCR assays, samples, standard curves, and normal controls must be run in triplicate.  The nice thing...the amount of DNA in your sample relative to your normal control - you can set up your calculations so that...RQ values used to call copy numbers should be as small as reasonably possible and set using the variance...though it does not require a standard curve or a normal control, it can take a few hours longer than qPCR...

...immunization has its drawbacks: it requires a camelid animal facility which can be expensive to gain access ...advantages of synthetic nanobodies Nanobodies are small (12-15 kDa) single-domain proteins that can bind...because it contains two large heavy chains and two small light chains that stick out to form the “arms” holding...a heavy chain antibody (HCab). Because of their small size, nanobodies can be synthetically expressed ...without the need to find a llama farm! To find an optimally binding sybody, you want to start out with a large...protocol goes through 3 major steps to select for optimal sybody candidates. Figure 2: Outline of the...