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...fluorophore from another within a fully stained sample. Figure 2 is an example of the emission spectra for ...another during spectral unmixing.     Figure 2: The full emission spectra for BV421 and BV711 on ...stained samples. If you are using conjugated dyes, it is critical that your controls and your sample antibodies...unmixing if your sample is stained with a PE/Cy7 antibody from a batch that is not the same as your single...allows the user to use the autofluorescence in a sample as a fluorescent marker. Given that the level of... varies, it is critical to include an unstained sample for each tissue type you are running in your panel...control(s) Compensation or “reference” controls are samples stained with a single fluorophore. They’re required...

... of permeabilization and membrane integrity (Fig. 2). For example, saponin interacts with cholesterol ...at too high a concentration.      Figure 2: Examples of different permeabilization strategies and... permeabilize a sample. But you can also use organic solvents after fixing your sample with a cross-linker...the point?  Fixation allows you to freeze your samples in a given state or point in time. You want to ...processes while maintaining the structure of your samples, the relationships between cellular components,... molecules and it is great for preserving your samples and maintaining cellular and tissue morphology....formaldehyde solutions.  Along with preserving your sample, cross-linking can block the epitope of some targets...

...managing to get together. If you all work at the same place, a 1-2 hour lunch on a regular schedule might work...The groups in these programs typically consist of 1-2 mentors and 3-5 mentees who meet together for an 8...Allows all group members to lead and learn at the same time Broadens the network of the participants Can...

... reduce the background staining (Table 2).      Table 2: Secondary antibody selections for a four-antibody...affinity binding, such as immunoprecipitations. Your sample species should also be considered when choosing...immunoglobulins naturally present in the tissue sample. There are species-on-species staining protocols...secondary antibodies that are all derived from the same host species and have been cross-adsorbed. The cross-adsorption...using mouse IgG2a and rat IgG1 antibodies in the same application, using a Goat anti-mouse IgG2a cross-adsorbed... species, and secondary antibodies raised in the same species that have been cross-adsorbed to reduce ...

...mean you have one copy of the gene; an RQ value of 2 means you have two copies, and so on and so forth....quantitative PCR. Expert Rev Mol Diagn. 2005 Mar;5(2):209-19. doi: 10.1586/14737159.5.2.209. PMID: 15833050...to a reference sequence present in the same amount in all samples (usually to a ubiquitously expressed,... reference sample with two copies of the gene (Ma, 2014.) As with all qPCR assays, samples, standard curves...housekeeping’ gene), and quantified relative to a 'normal' sample with known copy number set as the expression baseline...since you’re looking at the amount of DNA in your sample relative to your normal control - you can set up...number (RQ value) matches the copy number of your sample. In that case, an RQ value of one (or, more realistically...

...FRET signal back again. The Ting lab constructed 2 versions of this reporter to target the K9 (pcDNA3... 525.7570 (2015): 533-537. PubMed PMID: 26352475. 2. Bygren, Lars O., et al. "Change in paternal grandmothers...corresponding gene products due to the same region, but not the same exact genes, being improperly silenced.... This is how most cells in the body contain the same DNA but have very different structure and function...present without any DNA changes, and both involve the same region of chromosome 15. They both result from the...

...China, Korea, Japan, Brazil, India, and Mexico. Table 2: Addgene distributors Distributor Partnership...distribution after we partnered with them (Figure 2). In others, the distributor helped solve an issue...temperature for the duration of their journey. Figure 2: Annual distribution of items to countries with a ...that available and accessible weren’t always the same thing, shaped both Addgene’s founding and our growth...

...is unique compared to the rest of the genome and (2) the sequence is located just upstream of a Protospacer...pyogenes Cas9 variants with varying PAM sequences, 2) the use of Cas9 homologs derived from species other...Cpf1 Is a Single RNA-Guided Endonuclease of a Class 2 CRISPR-Cas System. Cell 163:759–771 . https://doi....and crRNA (or synthetic gRNA) are derived from the same species. Expanding the CRISPR toolbox The isolation...

...the reporter mRNA is labeled by PCP-mCherry (Figure 2). The 3’ UTR also contains a CAAX sequence to bind...PMC2746483 Find ribosome profiling plasmids at Addgene. 2. Halstead, James M., et al. “Translation. An RNA biosensor...highly useful, this technique requires extensive sample processing to avoid disrupting ribosome-mRNA complexes...

...target sites for editing. PAM sequences can be from 2-6 base pairs in length and follow the DNA sequence... Eric Perkins Mammalian genomic DNA is not just a 2-D linear molecule snaking through our cells’ nuclei...SpCas9) requires an NGG PAM to recognize the target. Osamu Nureki’s lab engineered SpCas9 (SpCas9-NG) with ...

...episode 2 "Organizing Literature" : I found out about Zotero here Dear Grad Student podcast episode 2 "Essential...and Linux.  Mendeley and Zotero have a lot of the same features, but this chart highlights the differences...

...immediate early gene expression. Genes Dev. 1988 Jun;2(6):718-29. doi: 10.1101/gad.2.6.718. PMID: 2843425...Targeted Gene Expression in Drosophila.” Genetics 186(2) (2010): 735-55. PubMed PMID: 20697123. PubMed Central... in Saccharomyces cerevisiae.” FEMS Yeast Res. 12(2) (2012): 197-214. PubMed PMID: 22129153. Additional...system to GAL4/UAS that functions in essentially the same manner, with LexA binding to the lexA operator (...desirable to control or look at multiple genes at the same time.  The 3 binary systems listed above are orthogonal...

...label) your sample (i.e. cultured cells or tissues).  Step 2: attach anchors to your sample that will link...so much. Created with biorender.com Steps 2-4 are not found in standard IHC/ICC protocols and you might...we just made the sample bigger? In ExM, and its various derivatives, biological samples are enmeshed in...the number of specific targets you can label in a sample. Super-resolution microscopy (SRM), a family of... its original size. Depending on the protocol, samples can be expanded to ~4-10x linearly! This expansion...gel components to polymerize. Step 3: digest the sample to allow for even expansion.  In some cases, labels... ExM process allows you to physically magnify a sample so you don’t have to optically magnify quite so...

...green squares, purple circles and blue triangles. 2) An antibody immobilized on a bead binds specifically...protein G, a novel IgG-binding reagent. J Immunol 133(2):969-974. Hjelm H, Hjelm K, Sjöquist J (1972) Protein... adjusted depending on the sample. It is important to remember that sample lysates will also contain proteases..., incubate the sample as you would in an IP using a nonspecific antibody from the same host species as... from the sample prior to beginning the IP.  During the IP, the target protein in the sample binds to ...parallel with both a sample known to express the protein of interest, such as a sample transiently transfected...out in one of two ways. Both methods utilize the same basic strategy: immobilize the antibody or antibody-target...

...linear fashion. This implies that reducing the power 2-fold does not reduce photobleaching by half. How exactly...prolonged time). Other illumination strategies, such as 2-photon-excitation, selective plane illumination, TIRF...This can differ between fluorescent proteins in the same spectral class (Cranfill et al, 2016). This non-...comparison of several fluorescent proteins of the same spectral class on the microscopy system and in the...

...11 and Cas13 are 1). non-specific RNA cleavage and 2). cell toxicity. Cas7-11 does not display collateral...Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell, 163(3): 759-771 (2015). 10.1016...isolated which target and cleave RNA through the same mechanism as traditional spCas9. If you are looking...Cas13 and has been proposed to be useful for the same applications (RNA knockdown, RNA editing, etc.)....

... Zetsche et al., Nat Biotechnol. 2015 Feb 2;33(2):139-42. doi: 10.1038/nbt.3149. Light-inducible ...and on your favourite signalling pathways (Figure 2). They are all available now at Addgene. 1 Lungu...20;19(4):507-17. 10.1016/j.chembiol.2012.02.006. 2 Guntas et al., Proc Natl Acad Sci USA 2015 Jan 6; ...Nihongaki et al., Chemistry and Biology. 2015 Feb 19; 22(2):169-74. High efficiency cloning for enzyme screening...inducible, gene knockdown with shRNA or shRNA-miR30, (2) tamoxifen-inducible CreERT2 gene deletion, (3) simultaneous...different fluorescent proteins, 8 constitutive promoters, 2 includible promoters, 3 polyA terminators and various...of plasmids required for each input/output system; 2) removing all chemically inducible promoters; 3) improving...

... you can read more about them in review articles (2–4). You can also take a look at my recent paper to...Development 118, 401–15 (1993). PubMed PMID: 8223268. 2. J. B. Duffy, GAL4 System in Drosophila : A Fly Geneticist...this mutant fly to one lacking the mutation in the same genetic background. In other cases, YFG or one of...

...that the signal to noise ratio can be increased from 2-fold (by CRISPRainbow) to 3-fold with CRISPR-Sirius...CNV) analysis of the U2OS cells.   Figure 2: CRISPR-Sirius tricolor labeling. (A) Diagram of sgRNA-Sirius...combination of MS2, PP7, or boxB sequences in the same sgRNA will generate additional colors. Image from...

...scientists have 1) the courage to make mistakes and 2) the integrity to recognize these mistakes quickly...She studies beta-cell function and failure in Type 2 Diabetes. Birmingham, AL, USA 33.5207°N 86.8025°W ...available to people who want to join the dialog and 2) remember that science should not be partisan: everyone...campus speakers thoughtfully reminded us to use the same critical lens on our scientific community as we ... nor would you mention my small error and at the same time pass over in silence his very large one” (http...exoplanets into a successful PhD thesis. We use the same atmospheric physics to understand exoplanets that...happy to see so many others come out who feel the same.  I was also incredibly impressed with how well ...