...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...

...Demonstration of PILATeS. Cell adhesion to a PILATeS-functionalized coverslip creates mechanical tension...reversible, making PILATeS suitable for live imaging with a time resolution of minutes.  Find PILATeS plasmids ...background signal when tested in more highly autofluorescent tissues like the intestine. Figure 3: ...antigenic differences between human, canine and simian isolates. J Gen Virol., 68 (Pt 11), 2769–2780. https://... happen within a few hours. In order to use the latest mScarlet tags, mScarlet3 and mScarlet-I3, Oliver...CRISPR/Cas9 workflow in M. abscessus. A) Sth1Cas9 mediates double strand break at the target site, which ...Bioluminescent reporting of mechanical forces with PILATeS By Emily P. Bentley Time to get flexible! Alex ...

...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...

...there, although other restriction sites (including other XbaI sites not preceded by GA or followed by ...occurring restriction modification systems that prokaryotes use to protect themselves from foreign DNA. The...digesting DNA. Why? Well, even though Dam methylation sites are not specifically associated with any restriction...systems, their sequences may overlap with restriction sites, inhibiting enzymes such as ClaI or XbaI, or, conversely...as DpnI require methylation at their recognition sites in order to efficiently cleave DNA. DpnI is often...mutation into your plasmid of interest by PCR generates mutated plasmids with no methylation (there are...commonly used in the lab do not have recognition sites that could overlap with a methylation site. The ...

...prefers ligation of Watson-Crick base pair substrates, it demonstrates significant activity on some mismatch-containing...experimentally-derived DNA Ligase fidelity data. Our work demonstrates that it is now possible to achieve 20+ fragment...amplicon form (shown above) are flanked by BsaI sites in an orientation that results in 5’ four base overhangs...pre-determined manner. If the original insert religates into its original vector, it will subsequently...target construct. Development of Golden Gate Assembly test systems Our research focused on three different ...phenotype upon growth on LB/Cam/X-gal/IPTG agar plates, indicating successful reconstruction of the coding...A final validation of the 5-, 12- or 24-fragment test systems was performed by setting up assembly reactions...

...results.  Replicates Replicates are another critical control in biological experiments. Replicates can be...of biological replicates such as multiple mice in each group and C, technical replicates or repeated measurements...specificity is to test the primary antibody against wild-type and knockout samples. In this test, wild-type ... control, you will be able to confirm that the testing protocols elicited the expected response and have...statistical power of an experiment. Technical replicates are repeated measurements of the same sample ...difficult to obtain meaningful results.  Biological replicates are distinct samples that are treated identically...mice, for example, it would be better to treat and test multiple mice with the drug rather than rely on ...

...serotypes and promoters to test in your AAV system and b) steps for testing and optimizing your serotypes...Transactivator Correlates with Long-Term Doxycycline-Regulated Transgene Expression in Nonhuman Primates after ...you know that, you’re ready to start selecting, testing, and optimizing three key variables of an AAV vector...titer, and promoter. Note that several steps in testing are effected by multiple AAV variables.    ...transduce your cells. In fact, we usually recommend testing more than one.  Most AAV serotypes have broad tropism...translate to in vivo efficacy. You’ll therefore need to test potential AAV vectors in your specific experimental...harm, your experimental goals when designing and testing your AAV.  Figure 2: Comparison of AAV ...

...13]:  Germ cells [14] Hepatocytes [15] Pancreatic β-cells [16] Intestinal tissue [17] Lung epithelial...22] Cardiomyocytes [23, 24] Smooth muscle cells [25] Skeletal myogenic cells [26] Chondrocytes [27...27]         Adipocytes [28] Keratinocytes [29] Photoreceptors [30] Otic hair cells [31] Neurons [32...reverts back to a pluripotent state, which then proliferates and is redifferentiated to a different cell ... from donors. Apart from fibroblasts, human keratinocytes from hair pluck, peripheral blood cells, and...transdifferentiation. Multiple cell types such as fibroblasts, hepatocytes, and pancreatic exocrine cells have been successfully...of induced pluripotent stem cells from human keratinocytes. Nat Biotechnol, 2008. 26(11): p. 1276-84. PubMed...

...recombineering substrates to avoid errors introduced during PCR. ssDNA Substrate ssDNA substrates can be generated...).  Shifting bacteria to 42˚C for 15 minutes quickly inactivates the repressor and allows for expression...Pro Tip: ssDNA substrates have a much higher rate of recombination than dsDNA substrates, so you should...modifications can only be made at restriction enzyme cut sites. The lambda red system is an alternative method ...within E. coli and is independent of restriction sites. The lambda red system is derived from the lambda...enzyme cloning where the genetic changes occur in a test tube. The donor DNA substrate only requires ~50 ...Beta: Beta protects the ssDNA created by Exo and promotes its annealing to a complementary ssDNA target ...

...and how each hypothesis will be tested with a specific statistical test.  Preregistration makes clear the...distinction between hypothesis testing work, in which a-priori hypotheses are tested with a new experiment; and...where reported methods may not include how many tests were conducted on how the results were analyzed....more credible than they actually are. Throwing out plates that don’t respond as expected, use of representative...encouraged to present that work using hypothesis testing statistical tools that generate a p-value.  As ...As an example, if you hypothesize that growth rates will be higher in treatment versus control, and you...rate, you report the results of all four of those tests and not just the subset of those that return a particular...

...contain a mix of cells including granulocytes, lymphocytes, and monocytes. Due to differences in size and...separate into distinct populations of monocytes, lymphocytes and granulocytes in a plot of forward scatter (FSC...and fluorescent light sources. Visible light illuminates each cell as it passes through and scatters in...intensity of events. Since intensity positively correlates with expression, the further the shift of a cell...staining is quicker than indirect staining and eliminates potential non-specific staining that may arise...through the cell membrane. Formaldehyde fixation creates cross-links between lysine residues thus preserving...subset of cells. During FACS, the sample stream oscillates to generate droplets that are charged as they...

... or decreased off-target activity across the sites tested when compared to eSpCas9(1.1) and SpCas9-HF1...editing can result in unwanted changes at non-target sites. Means to decrease these so-called “off-target effects...activity of the Cas9 nuclease itself. Design and testing of eSpCas9 Looking at the structure of the Cas9...However, this is not always enough to keep off-target sites from being cut. Slaymaker et al reasoned that, if...separation afforded by gRNA binding at non-target sites would theoretically decrease off-target cutting....throughout the groove in 32 separate Cas9 mutants. When tested for their ability to cut the EMX1 locus in human...went on to combine the best of these mutations and tested the combination mutants for their on-target and...

... DNA sequences located between two loxP sites. The loxP sites are present at both ends of a gene in the...include Swiss Webster and Harlan (Hsd) National Institutes of Health (NIH) Swiss. Genetically engineered...embryonic stem cells. Non-homologous recombination incorporates this DNA randomly into the mouse genome. In ...also included to select for successfully modified zygotes or embryonic stem cells. Modified embryonic stem...vector containing homologous ends to the breakpoint promotes homologous recombination of the targeting vector...undergone genetic modification that alters or eliminates the expression of a particular gene. Targeting...because non-homologous repair is a random process, zygotes edited using the same nuclease may not result in...