We narrowed to 513 results for: gats

...a clear separation between positive (blue) and negative (gray) droplets. The no template control wells...The ddPCR software uses the ratio of positive to negative droplets is used to calculate the concentration...getting a clean separation between positive and negative droplets with these parameters, here are a few...leading to greater separation of your positive and negative droplets. No more than 50 cycles is recommended...but you can go as low as 1C/s. Increasing the elongation time to 2 minutes and the denaturation time to... Witte, Anna Kristina, et al. "A systematic investigation of parameters influencing droplet rain in the...

...Plasmids from the Tsuyoshi Nakagawa lab are binary gateway vectors and can be used for promoter swapping and... al., 2007). Alternatively, if you prefer Golden Gate assembly, there are a series of plasmids from the...transformant with a single insertion because it will segregate 3:1 in the T2 generation. You also have no control...genotype in T1 than T2. Your transgene should not segregate in T1, but it probably will in T2. Laura Lee...Dinneny, J. R. (2013) ‘A robust family of Golden Gate Agrobacterium vectors for plant synthetic biology...PMC4426443. 4. Nakagawa, T. et al. (2007) ‘Improved Gateway Binary Vectors: High-Performance Vectors for Creation...

...gel and purify the digested vector. Step 4: Ligation Ligate the annealed oligos into the digested vector...blog post was written by Dr. Kutubuddin Molla, investigator at ICAR-Central National Rice Research Institute...radiodurans (Dra2TnpB) recognizes the TAM sequence 5′-TTGAT-3′, while TnpB from Deinococcus geothermalis (Dge10TnpB...the 20-nt Guide Sequence Identify a TAM site (5′-TTGAT-3′) in your target gene. Choose a 20-nt sequence...account: @Kutub_joy). Dr. Kutubuddin Molla is an investigator at the ICAR-Central National Rice Research Institute...

...the cytoplasm. That is, unfortunately, some investigators realize too late that they have missed the impact...confound quantitative imaging experiments or even negatively impact cells (see Figure 1). When behaving properly... properly, FPs enable researchers to investigate the localization and dynamics of fluorescent protein ...environment where it will localize [4]. Most investigators are surprised to learn that FPs are often poorly...overestimation of fusion protein activity. We encourage investigators to characterize FP-fusion proteins functionally...chloroplasts, the extracellular milieu, and gram negative bacteria periplasm. Efforts to tag endogenous ...

... mutated to a negatively charged residue, aspartate or glutamate, imparting the negative charge associated...smaller, and carry only a single negative charge as opposed to a double negative charge in the phosphate group...charges this tRNA with phosphoserine A specialized elongation factor that allows proper extension and translation...Sep-tRNA synthetase, and EF-Sep – an optimized elongation factor that accepts Sep-charged tRNAs during ...

... recombination has not occurred. This powerful negative selection system ensures high editing efficiency.../gRNAs are easily inserted using BbsI and Golden Gate Assembly. Either plasmid can be also linearized ...repress transcription by physically blocking the elongation complex from binding the DNA or extending the...activation of the system. Mobile CRISPRi for Gram-negative and Gram-positive bacteria Jason Peters, Oren ...different genetic systems, targeting either Gram-negative bacteria or Gram-positive firmicutes, to bring...introduced (Tn7 in Gram-positives and ICEbs1 in Gram-negatives) but share the common CRISPR-dCas9 system for ... Sharp JD, Xu R, Prisic S, Husson RN (2016) Investigating essential gene function inMycobacterium tuberculosisusing...

...electric current applied to the gel causes the negatively charged proteins to migrate toward the positive...binding.  Whenever possible, include positive and negative controls. Positive controls typically include ...relevant levels (not overexpression systems) while negative controls are those that do not naturally express... on the membrane the antibodies are typically conjugated to an enzyme, such as horseradish peroxidase ...protein of interest, is not labeled. Instead, a conjugated species-specific secondary antibody is used to..., take care to include the proper positive and negative controls, choose antibodies that are validated...

... of CRISPR technologies, one might incorrectly relegate RNAi to history. The straightforwardness of RNAi..., gene knockout strategies cannot be used to investigate essential genes. Historically, gene silencing...present at the first stage of an RNAi-based investigation. Creation of the RNAi trigger requires a few... which allows easy creation of long hpRNA with Gateway-based cloning and straightforward positive selection...assembly of intron-spliced hpRNAs through Golden Gate cloning. Artificial micro RNA and short hairpin ...from Worcester Polytechnic Institute where he investigated how plants achieve polarized cell growth in ...

...Overexpressed proteins also have the potential to form aggregates and may be toxic to the cell. Here comes the ...Initially, Tanenbaum et al. observed some GFP aggregation, which they reduced by using superfolder GFP ...expression levels of SunTag constructs would avoid negative effects on cell physiology. Having seen that mitochondrial...Since the v4 system does not display protein aggregation, it’s recommended for most imaging applications...with SunTag. v1 SunTag exhibits some scaffold aggregation (v4 Suntag does not.) It’s up to you to determine...

...resistant to a toxin), whereas negative selection would be the picking up of a negative trait (i.e. the plasmid...below we focus on positive selection; however, negative selection techniques can be used in conjunction... polypeptide synthesis at 80S; inhibits chain elongation 100-800 ug/mL Hygromycin B hygB HeLa, NIH3T3... polypeptide synthesis at 80S; inhibits chain elongation. 50-500 ug/mL Puromycin pac HeLa, 293HEK, Jurkat...

...Distribution Please indicate which strain Addgene should propagate your plasmids in: DH5α, NEB Stable, or ccdB Survival...Survival.­ Whenever possible, we propagate plasmids in the standard cloning strain DH5α. Please indicate...indicate if your plasmid cannot be propagated in DH5α. For plasmids with highly repetitive sequences (which...For plasmids containing the ccdB gene, such as Gateway vectors, we recommend the ccdB Survival strain....correct. You can easily update the information by navigating to the previous pages using the links at the ...deposit, associate the plasmids with a Principal Investigator (PI), provide us with your shipping address ...

...Inducible promoters can be regulated by positive or negative control. Positive inducible In the OFF state, ...turning it ON and initiating transcription.     Negative inducible In the OFF state, the promoter is inactive...most commonly used prokaryotic promoters is the negative inducible pLac promoter. This promoter requires...is found in many bacterial expression vectors. Negative inducible promoter pBad is another popular prokaryotic...pGGA008 (AlcA) and pGGC011 (AlcR) from red flame GreenGate kit Steroid-regulated LexA promoter XVE (synthetic...

...paired end complex The paired end complex then ligates compatible DNA ends together, thus repairing the...need to be processed. Processing occurs prior to ligation as incompatible DNA ends interfere with that step...that function to make ends better substrates for ligation. Although we do not describe these steps here,... sequence (“microhomologies”). NHEJ doesn’t obligatorily introduce indels. Given the end structure of...Hendrickson EA, Ramsden DA (2014) The fidelity of the ligation step determines how ends are resolved during nonhomologous...

... to be directly conjugated to a reporter or you need a light-chain specific conjugated secondary antibody...find these in a Fab fragment format. Reporter-conjugated anti-IgG Fab fragments make good secondaries ...as a block in similar scenarios. By applying unconjugated Fab fragments that are the same species as your...chain on a Fab’ can act as a convenient site for conjugation.  Finally, Fvs are the most amenable to recombinant...

... activity. 2. Studying negative feedback loops of signaling pathways Negative feedback loops are a common...Additionally, dimeric Dronpa is less prone to aggregation in cells compared to the tetrameric Dronpa it...pulses of kinase activity. Zhou et al studied the negative feedback on endogenous MEK1/2 phosphorylation ...suggesting PP1/PP2A is responsible for this MEK1/2 negative feedback. 3. Studying signaling pathways in vivo...

...homogenate and shaken. After a quick centrifugation (not ultracentrifugation!), the layers of phenol and chloroform...bacteria, keep samples cold and work quickly to mitigate RNA degradation. Make sure to use DEPC-treated...out to develop a protocol that skipped the ultracentrifugation altogether. Chomczynski and Sacchi showed... TRIzol®, insoluble material is removed via centrifugation and the supernatant is extracted with chloroform...

...they can’t be genetically tagged and instead are conjugated to reporters (i.e. fluorescent dyes, HRP, etc...single domain antibody. To create their RANbodies, Yamagata et al used previously published nanobody sequences...they produce the same signal, so only one HRP-conjugated RANbody can be used at a time. Samples can be...RANbodies? Great! All of the RANbody plasmids from Yamagata et al can be found here. Can’t find a RANbody ...the comments below. References Sanes, J.R., & Yamagata, M. (2018). Reporter-nanobody fusions (RANbodies...

...are 11 different genes cloned into plasmids to investigate for each of these planarian species. These plasmids...wildtype locomotion.   Create your own plasmids to investigate other genes in planarians! For those who are ... performing their own molecular cloning and investigating genes not in this collection, they may clone...stowers.org and if you make new plasmids for investigating different planarian genes, we encourage you ... other classrooms and researchers can easily investigate in planarians. References 1. Alice Accorsi, ...

...initial timer was a tetramer with a propensity to aggregate in cells (Tsuboi et al.), limiting its use. Nonetheless...2000, researchers have realized how problematic aggregation-prone tetrameric fluorescent proteins can be....functionality are all possible consequences of FP aggregation, as Erik Snapp’s blog post shows. To avoid these...developed monomeric FP timers with less propensity to aggregate. These monomeric FP timers (mK-GO from Tsuboi ...Addgene Blog Learn More About Fluorescent Protein Aggregation Catch Up On Your GFP Basics Get Advice on Choosing...

...they are negative-going sensors (with fluorescent signal at baseline voltage), the investigators found that... or membrane protein (Plasmid #141386-LV [+M], negative control) were immunoblotted against Anti-SARS-... Figure 4: Structure of Voltron or Voltron2 conjugated to a JaneliaFluor dye emitting at 525 nm. Image...fluorescence is tunable by changing the dye that becomes conjugated with its HaloTag domain.  Find Voltron2 plasmids...