...location and orientation of the loxP sites: Inversion: If the loxP sites are on the same DNA strand and are...region of DNA between the loxP sites is reversed.   Deletion: If the sites face in the same direction, ...If the sites are on separate DNA molecules, a translocation event is generated at the loxP sites. How can...limitation of using loxP sites is the inability to tightly control which loxP sites recombine if more than...GtATACAT). These variant lox sites undergo recombination with other sites of the same type, but are not...inter-molecular recombination at the loxP recognition sites. A key advantage of the system is that Cre acts ...experiments. LoxP (locus of X(cross)-over in P1) sites are 34-base-pair long recognition sequences consisting...

...between the sites (Fig. 1B below). Alternatively, if an attB-attP pair is anti-aligned (sites facing opposite... their recognition sites, or by excising one or multiple of their recognition sites from the register..... Figure 1D below demonstrates an example of a register that enters distinct states when treated with ...recognition sites, terminators, promoters, and genes) were built with flanking primer binding sites (designed...registers. The primer binding sites can also be used to design primers to read states by qPCR or Sanger sequencing...that recognizes its own attB and attP recognition sites on the register and catalyzes their recombination...state of the RSM. If an attB-attP pair is aligned (sites facing the same direction) on a register, then the...

...Rep and Cap shown in the figure on the right facilitates the viral pseudotyping discussed below. Overview...As part of its lysogenic cycle, wild-type AAV integrates into the host genome at a specific site, AAVS1...than wild-type AAV to integrate at non-homologous sites in the genome, and do so at about a 0.1% frequency...serotypes are denoted using a slash, so that AAV2/5 indicates a virus containing the genome of serotype 2 packaged...spontaneously annealing, upon infection, which eliminates the requirement for host cell DNA synthesis. ...

...recognizes FRT sites and initiates site directed gene recombination between those sites. FRT sites and loxP ... of target sites that differ in their sequence. Recombination between the two target sites with identical...and the interior target sites. In a second step, the two compatible target sites are cleaved out, leaving...loxP sites differ at the nucleotide level but share an overall structure of 13 base pair palindromic repeats...bp asymmetric core. Recombination between these sites can lead to excision, inversion, and translocation...translocation of DNA in a similar fashion as between loxP sites (For details see Cre-lox blog post). Optimizing ...systems in your experiments. Mutated FRT and loxP sites in Flex Vectors – inversion only once All recombination...

...considerations for behavioral testing As a general consideration for behavioral testing in a laboratory, a high... some time to adapt to a testing apparatus. General stress levels of the test animal may also be reduced...AAV5 targets both neurons and astrocytes, and AAV4 targets mostly astrocytes. Lentiviral vectors are pseudotyped...weeks before the behavior test. This time window between injection and testing will vary depending on whether...influence on the outcome and cause variability in the testing group. For example, if an experimenter picks up... will likely move around faster in the following test and skew results measuring locomotion. Mice may ...reduced by daily handling for a few minutes by the experimenter several days preceding the experiment. Handling...

... with three coordinates: chromosome, start, and stop. For instance (chr3, 1, 10) indicates a stretch of...notation works? The + operator denotes intersection and the – operator denotes set difference. That’s the ... critical NANOG binding sites: 1. Find Enhancers that have NANOG binding sites 2. Design a CRISPR screen...all of the DHS sites from 125 different human cell types from the ENCODE Project. DHS sites are the most...as a kind of “GPS for the genome” to compute coordinates for genetic elements like protein, RNA, and DNA...DNA (Table 1). As with GPS in the real world, coordinates alone aren’t very useful. We’ll need something...come in. They help us to transform raw genomic coordinates into meaningful experiments. Table 1: An emerging...

...bits (1.375 bytes) of information. This leads to 2048 (211) possible combinations of states. Each of the...protein binding sites and 24 copies of the MS2 bacteriophage coat protein binding sites to tether many ...reporter proteins that can be used to test diverse secretory routes in various conditions. The plasmids ...enzymes, such that the ligation regenerates the original restriction sites for further iterative cloning...2xNLS) and c-terminal (Cas9(C)-2xNLS) fragments facilitates target DNA cleavage upon co-expression and spontaneous...which act irreversibly on their cognate attB/attP sites with no cross-talk. These pairs are closely related... in various combinations, while the cognate att sites are supplied as individual reporter constructs or...

...cutting at mismatched guide sites, but not necessarily binding to these sites. Thus, these enzyme swaps ... ChIP-Seq, where only DNA sites bound by Cas are sequenced, has been tested with some success. The major... sequencing Sites with high sequence similarity to the gRNA can be sequenced. These sites can be selectively... this often means DNA cleavage at unanticipated sites. While off-target effects are nearly impossible ...or is engineered not to cleave DNA and instead activates/represses gene expression by various mechanisms...words, they can sometimes localize to unintended sites with sequence similarity to the on-target site. ...When a CRISPR-Cas system localizes to unintended sites and performs its programmed function there, this...

...behavioral tests.    Forced swim test The forced swim test has been used since the 1970s to test the efficacy...general considerations for performing behavioral tests, but now we’ll focus on how viral vectors have changed...locomotion, anxiety, and depression.  Locomotion tests To study and quantify locomotion, scientists monitor...Such a behavior can be monitored in an open field test (Alpar et al., 2018). Locomotion can also be used...therapeutic approach against drug addiction. Anxiety tests Rodents have a natural preference to stay in dark...neurons turns on overexpression of IL-1 beta. They tested the mice in a series of behavioral tasks. One of... of antidepressant treatment in rodents. In this test, an animal is placed into a tank of water with no...

...the effect of the independent variable you are testing. Importantly, they demonstrate the functionality...associated with the independent variable you are testing and not some other unintended factor. Control plasmids...luciferase) that is either co-transfected with the test plasmid or transfected into a separate well of your...Regardless, the amount of reporter protein activity correlates to both the amount of DNA transfected into the... Plasmid B (as internal control) This result indicates that the transfection was not successful due to... and from different preparations of the purified test plasmid expressing the independent variable. There...technical and biological. In general, technical replicates can be thought of as "plate controls". They are...

...generated by the nickases; D10A creates 5’ overhangs and H840A creates 3’ overhangs in a PAM-out design...problem. D10A nickase outperformed both WT Cas9 sites tested by a wide margin, exhibiting >20% repair efficiency...Optimize your spacing D10A: nick sites separated by 37-68 bp H840A: nick sites separated by 51-68 bp Use...CRISPR nickases improve homology directed repair rates, and share their design rules for your next CRISPR...as examples for this post. The D10A mutation inactivates the RuvC domain, so this nickase cleaves only...Conversely, the H840A mutation in the HNH domain creates a non-target strand-cleaving nickase. Instead of...Mollie Schubert and Shuqi Yan designed experiments to test nickase preferences using HEK293 cells. They started...

...widespread testing is crucial in controlling this pandemic. Currently, the CDC uses RT-qPCR tests to diagnose...100% negative prediction rate. It is also the fastest test that has been developed so far.  A similar method... Highly similar to the SHERLOCK diagnostic test, this test varies in the sequence of steps and the Cas...and to do so, the WHO recommends one strategy: testing, tracking, and social distancing. With countries...diagnose COVID-19 and some serological tests to determine past exposure. However, the limited availability...general schematic of CRISPR/Cas based COVID-19 testing methods.   Rapid detection of 2019 novel coronavirus...:10-100 copies of RNA per microliter Time: ~40 minutes Equipment required: 37°C heat block and 62°C heat...

...expression piggyBac Excisible Medium Medium Generates transgene-free and vector-free cells Genome integration...mutations Adenovirus Non-integrating Low Medium Generates transgene-free and vector-free cells No genomic... Sendai Virus Non-integrating Medium Medium Generates transgene-free and vector-free cells No genomic...Plasmid Non-integrating, usually Medium Medium Generates transgene-free and vector-free cells Limited genomic... EBNA1 episome Non-integrating Medium Medium Generates transgene-free and vector-free cells No genomic... Minicircle Non-integrating Medium Medium Generates transgene-free and vector-free cells No genomic..., criteria used, etc.) can effect reprogramming rates and such a number maybe misleading. Integrating ...

...higher mutation rates than they’d previously reported in protoplasts (15% and 9% of target sites.) How many...efficient tRNA processing is common to prokaryotes and eukaryotes. Though the plasmids made by Xie et. ...RNAs (gRNAs) are commonly packaged in 400-500 bp cassettes containing the RNA pol III promoter, gRNA and ...and pol III terminator. These relatively large cassettes (considering the gRNA itself is ~100 bases) limit...cleave 4 gRNAs separated by 28 base Csy4 recognition sites from a polycistronic transcript. Although this method... or pre-tRNAs are cleaved by RNase P and Z in eukaryotes (E in bacteria), and only a few elements of tRNAs...recognition and cleavage. This sequence flexibility indicates that gRNAs could be incorporated into these constructs...

...Subscribe to the Addgene blog to get all the latest hot plasmid updates!     Tol2 Gateway-compatiable toolbox...vectors that have standard cloning sites or gateway compatible cloning sites for easy cloning of your genes...variations in gene expression from these polycistronic cassettes could lead to comparable experimental results....compared different variants of the polycistronic cassettes and discovered reprogramming discrepancies, which... Klf4 isoform (KLF4s or KLF4L). Polycistronic cassettes that contained KLF4s had overall diminished KLF4...global gene expression patterns. Polycistronic cassettes that contained the KLF4L isoform had more robust.... The polycistronic piggyBac transposon vectors tested in this work are available at Addgene. Kim, et...

...intronic regions and two repair templates in order to insert two loxP sites. Since two double-strand breaks... Single-stranded DNA repair templates Short single-stranded DNA templates have been shown to improve the...2 loxP sites to intronic regions 0.5 - 0.8 kb apart. In the example below, we’ll add loxP sites for conditional...disrupting splice donor/acceptor sites, Miura et al. (2018) recommend targeting sites at least 100 bp away from...place the loxP sites precisely at the cut site for each guide. This design also separates the gRNA target...services or kits, including the IDT Megamer service tested by Quadros et al. When delivering the ssDNA template...kb or less. Luckily, most of the commonly used cassettes, such as fluorescent proteins (EGFP, mCherry etc...

...availability of enhancer sequences in different chromatin states. Alexander Stark and colleagues at the Research...self-transcribing active regulatory region sequencing) that tests enhancer sequences outside of their endogenous chromatin...Synthetic and basic biologists alike often need to test multiple expression systems before they achieve ...bacteria, the process of cloning, combining, and testing these different parts can be quite arduous. The... lab generated 5 common 30bp overlap sequences (Sites #1-5). Each overlap site encodes a unique restriction...restriction enzyme site and unique primer sites for more rapid screening by restriction digest and Sanger ...allows scientists to easily design, generate, and test constructs composed of complex genetic elements....

...go over some of the common conjugates you may encounter and how conjugates and antibodies come together...enzyme conjugates are Horseradish Peroxidase (HRP) and Alkaline Phosphatase (AP). Various substrates for ...together. Common conjugates in the lab Fluorophores Fluorophores allow you to detect your antibodies using.... We also touched on fluorophores as antibody conjugates in an earlier post, where you can find some additional...biotin and its use in molecular biology. Other Conjugates to Know It would be overwhelming to try to cover...cover all of the possible antibody conjugates (especially if you start to consider clinical applications...potential. You will find antibody-oligonucleotide conjugates used in assays like proximity ligation, immuno-PCR...

...transfection into plates containing selection agent at different densities, then dispose of the plates that do ...to cloning rings, dilution cloning into 96-well plates, is extremely time- and resource-consumptive, since...cell can give rise to single clones, and thus many plates must be examined for single clones and then handled...can pick 48 clones from a 10 cm dish in 15- 20 minutes- a far more rapid pace than using cloning rings...12-48 individual colonies by (carefully) holding plates up to the light, where they appear as opaque dots... achieved by splitting transfected cells into 3 plates at different densities for selection). A 2% agarose...are used). This solution is capped, swirled and tested for the correct temperature by placing on the wrist...

...reinforces behavior (activation of the circuit promotes subsequent, repeated activation), this is an approximation...perceives through taking a drug. The ideal behaviors to test reinforcement are ICSS and place preference.   Historically...neuronal cell-type and temporal precision. In ICSS, a test chamber is used in which a nose-poke at a particular...reinforcement but is considerably simpler than ICSS. The test chamber is divided into two halves that are distinguished... certain technical considerations still apply. Testing stimulation First, In lieu of electrophysiology...targeted neuronal population prior to behavioral testing. One method to determine this is to examine expression...be low or highly variable without the behavioral test. Another readout may be necessary to prove inhibition...