...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.)....

...specific control of activity. Read More CRISPR Class 2 C lustered R egularly I nterspaced S hort P alindromic...plasmids. Plasmids within a given library have the same backbone, but they express or target different genes...

... 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...

... 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...

...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 ...

...expressed as a fusion with your favorite protein and (2) a synthetic fluorophore-contianing “ligand” that ...cell 58.4 (2015): 644-659. PubMed PMID: 26000849. 2. Grimm, Jonathan B., et al. "A general method to improve...removing excess dye from the sample, which is particularly important for samples where washing is difficult...reason is something called “the photon budget”: each sample has a limited number of fluorophores and each fluorophore...of information one can extract from a biological sample is wholly dependent on the photon budget and pushing... desperate to extract a few more photons from a sample. Chemical fluorophores can be substantially brighter...

...Table 2: Lab strains of E. coli Strain Natural resistance Primary Use Genotype ccdB Survival 2 T1R ...resistance to tetracycline Additionally, Table 2 provides a quick reference for some of the popular...convoluted due to researchers sharing and renaming samples throughout history. It was likely isolated in 1918...

...sites would confound your experimental results and 2) perform targeted off-target measurements at those...experiments where only localization of the Cas occurs. 2 gRNA approach Off-targets that come from mutations...perform downstream characterization of, isolating 2 or 3 distinct clones to perform key experiments on...If you are performing CRISPR library screens, the same principle holds true - the higher the number of ...

..., and the desired gene (transfer vector plasmid), 2) the HIV-1 genome containing all viral genes with ...protocols that have been described (protocol 1, protocol 2, protocol 3) to find the one that will give you the...PubMed PMID: 3785217. PubMed Central PMCID: PMC367857. 2. Naldini, Luigi, et al. "In vivo gene delivery and...are working with proto-oncogenes or with human samples that have not been tested for HIV.  If not using...ribosome entry site (IRES) and expressed under the same promoter. Alternatively, your gene and the marker...

...of ~75% in response to the magnetic field (Figure 2); they named this variant MagLOV. Andrew was thrilled...didn’t need any added cofactors.      Figure 2: Fluorescence intensity and ΔF/F of MagLOV, a variant...fluorescence when a small magnet was passed over the sample (Frank Hayward et al., 2024). The fluorescence ...intensity decreased whenever the magnet approached the sample (by less than 1%) and recovered after the magnet...expressing MagLOV while a magnet is waved under the sample. The imaged area measures approximately 82 mm across...Matsuoka, R., Kimura, S., Miura, T., Ikoma, T., & Kusamoto, T. (2023) Single-Molecule Magnetoluminescence...

... tropism and axonal transport of AAV serotypes 1, 2, 5, 6, 8 and 9 in the mouse brain. PLoS One. 2013 ...PMID: 24086725. PubMed Central PMCID: PMC3785459. 2. Krashes MJ, Koda S, Ye C, Rogan SC, Adams AC, Cusher...systems typically express Cas9 and a gRNA from the same plasmid. LentiCas9-blast virus can be used to generate...

...per cell increases 4-5 fold (for pUC plasmids) or ~2 fold (for p15a, R6K, and ColE2 plasmids) as cell division...biotechnology 36.4 (2018): 352. PubMed PMID: 29553576. 2. Kittleson, Joshua T., Sherine Cheung, and JChristopher...other hand, using a constitutive promoter in the same background resulted in a 20-fold variation of gene...

...connectivity to other data sources used in research; (2) connectivity to other tools used in research; and...by making it easy to get data out of the ELN; and (2) facilitate inter-operability with other research ...Github and Gitlab, and science-specific tools, e.g. sample management apps, colony management software, and..., not all of them support reproducibility in the same way. When choosing an ELN for your research, be ...commercialization community in greater Boston, while at the same time keeping one foot on the other side of the pond...

...estimate transformation efficiency (Figure 2).    Figure 2: Example of a serial dilution to estimate...library from Addgene, they’ll need to amplify the sample for further downstream applications. As individual... all of the bacteria can grow and compete in the same container, making it easier for a smaller, recombined...

...nonsense-mediated decay. Although Nelson et al. observed only 2% genome editing in one experiment, they found the ... cells throughout the body, especially stem cells 2. Remove any immunogenicity of the AAV vector Once ...Science. 2015 Dec 31. doi:10.1126/science.aae0169 2. Long C, Amoasii L, Mireault AA, McAnally JR, Li H...slightly different methods, they each observed the same positive phenotypes in cardiac and skeletal muscle...

...form or as ready-to-use lentivirus. Figure 2: dCas9-Tet1 demethylates cytosines at promoters and...pX330a dCas9-LSD1 for targeted removal of H3K4me1/2 and H3K9me2 histone modifications. Like the TALE-LSD1...approach for targeted DNA demethylation. Cell Discovery 2: . https://doi.org/10.1038/celldisc.2016.9 Find plasmids...activate transcription. A lentiviral vector with the same modifier, Fuw-dCas9-Tet1CD, is available from Rudolf...

...residues, which can be found throughout the antibody. 2) Endogenous cysteines, such as those that make up ...Detecting antigens by quantitative immuno-PCR. Nat Protoc 2:1918–1930. https://doi.org/10.1038/nprot.2007.267 ... antibody will have been verified to perform the same as its unconjugated version, but if you find yourself...

...et al., 2020). Genomic targets require an upstream 2-bp PAM sequence for recognition by QCascade; although...cells of the plasmid after integration. Figure 2: (A) Streamlined VchINT expression constructs. (B)...seven required genes and the CRISPR RNA under the same promoter, and a donor plasmid (pDonor) encodes the...immunity, whereby multiple proximal insertions with the same transposon exhibit reduced efficiencies (Vo et al...

...retain their individual activities when separated (2), and that DBD and TAs from different systems could... Enzymol. 1983;100:293-308. PubMed PMID: 6312261. 2. Keegan, L., Gill, G., and Ptashne, M. Separation ...libraries of bait and prey against each other at the same time in order to try and access the entire interactome...the protein partner or the UAS/reporter gene, the same bait and prey libraries can be screened using both...

...with neuron-specific expression of dCas9-VPR (Figure 2), a popular Cas9 activator developed by the Church...ubiquitous ones such as EF-1α and CAG. Figure 2: Dual lentivirus system with ubiquitous expression... Gene isoforms refer to mRNAs generated from the same gene but with different untranslated or coding regions...transcriptional start sites, all of which yield the same BDNF protein. To demonstrate the specificity that...

... pCRISPomyces-2 contains Cas9 and a gRNA cassette. The simpler system of pCRISPomyces-2 displays a higher...and a subsequent curing protocol that requires only 2-3 hours incubation. E. coli and T. citrea scarless...four major components including (1) a CRISPR RNA, (2) four proteins forming the QCascade DNA-targeting ...https://doi.org/10.1038/nbt.3043 Bikard D, Jiang W, Samai P, Hochschild A, Zhang F, Marraffini LA (2013) Programmable...

...different activators exist, including the multi-plasmid SAM system. Select Expression System To use CRISPR, you...either (1) a lack of gRNA and/or Cas9 expression or (2) a lack of efficient target cleavage in cells expressing...efficacious than gRNAs containing a C nucleotide at the same position in spite of being a perfect match for the...

...blood are reprogrammed immediately after collection [2]. PBMCs display unique epigenetic signatures that ...PMID: 27882265. PubMed Central PMCID: PMC5118044. 2. Kim, Y., et al., The Generation of Human Induced ...blood sample, in the best-case scenario [7]. In the clinical setting where patient blood samples are limited...grade hiPSCs from easily accessible patient blood samples. All pCXLE vectors listed below in Table 1 are ...

...days before the deadline isn’t going to be much use. 2. Originality is essential for grant success The two... it’s important not to throw in the towel if 1 or 2 funding bodies say no. If possible, get feedback from...institution). Likewise, if you’re staying in the same lab where you have been working for the last few...

...from animals or hybridomas.   Figure 2: (Left) Figure 2: (Left) The light chain and heavy chain genes...antibody expression. If starting from a protein sample (for instance, affinity purified antibodies from...

...is funded have been well documented over the past 2 years (Alberts et al, PNAS 2014; Taylor, eLife, 2016...PMID: 24733905. Pubmed Central PMCID: PMC4000813. 2. Taylor, Justin Q., et al. “Avoiding a lost generation...postdocs at institutions, will be challenging. At the same time, there is much opportunity to make headway ...

... both substantial merit and national importance; (2) The foreign national must be well positioned to advance...outstanding achievements in your specific academic field, (2) that you have three 3 years of research experience..., Casseus Law suggests a mix of experts including 2 dependents –people who have worked with you i.e. your...working in October of the same year. The Industry H-1B visa is not the same as an academic H-1B visa and...individually, as a judge of the work of others in the same or allied academic field Evidence of original scientific...

...everything but the CD8+ T cells (Figure 2). Even better, we can use the same fluorophore for the three lineage...     Figure 2: Dump gating labels all unwanted cell lineages with the same fluorophore to easily...FACS achieves greater purity of your sorted cell sample than MACS. An effective strategy to sort out your...different while the permeabilization solution can be the same). Examples of such targets include transcription...undesired cell populations. Dump gating uses the same color for different markers, making it easy to gate...

...required components are 1) a SMASh-tagged protein and 2) asunaprevir (small molecule). dTAG The degradation...gone through an additional round of evolution (AID-2) to reduce some of the leakiness associated with the...the general concept of the system has remained the same. An obvious drawback of the system is that it requires...

...engineering methods have their own pros and cons (2). The NgAgo DNA guide is inexpensive and can be 5’...Nature biotechnology (2016). PubMed PMID: 27136078. 2. Burgess, Shawn., et al. "Questions about NgAgo." ... to deliver NgAgo ingredients into the cell, the same method used by Gao et al, 2016 (1). View the Complete...

...from GFP and the mutation definitions found in Table 2 may not apply. Table 1: The specific mutations comprising..., V163A EBFP F64L, S65T, Y66H, Y145F   Table 2: Functional role of specific mutations in GFP derivatives...that of the gene of interest, or expressed from the same transcript as the gene of interest, but after an...

...the chromosome, ending replication.   Figure 2: Prokaryotic chromosomal replication. Adapted from...Microbiology and Molecular Biology Reviews: MMBR, 62(2), 434–464. https://doi.org/10.1128/MMBR.62.2.434-464.1998...machinery in order to replicate—but not always to the same extent. As described in our previous Origin of Replication...proteins, it can be regulated indirectly via the same mechanisms that affect DnaA and IHF. RepA also controls...

...faster when using a dual guide system.   Fig. 2: Size screening for large deletion events.   ... Biol 17: https://doi.org/10.1186/s13059-016-1012-2 Find more CRISPR Resources at Addgene: Read our CRISPRGuide... as a proxy for your edit of interest. Using the same gRNA and donor, you can deliver a silent mutation...to compare the sequencing reads from your edited sample to an untreated population. Software such as CRISPResso...

... a western, proteins are: (1) separated by size, (2) transferred to a membrane, and  (3) detected using...expensive system-specific sandwiches.   Figure 2: The western blot dry apparatus includes a stack of...thiols, to cleave disulfide bonds. Load the samples, one sample per lane, onto the top of a resolving gel...all of the proteins in a sample migrate through the stacking gel at the same pace and enter the resolving...consistent size and density present in all samples. Uneven sample loading or incomplete protein transfer ...Figure 1: Overview of the Western blot process. The sample, a mixture of protein is separated in a gel, transferred...is the first step of a western. To prepare the samples for SDS-PAGE, measure the protein content and normalize...

...another. You’ll need to validate that points 1 and 2 hold true under your specific experimental conditions...when (1) other strategies may not be feasible and (2) large-scale proteomic and transcriptomic datasets...antibody signal in the retinal sample, but little to no signal in the liver sample, which you do (C), so your...epitopes on the same target reduces the likelihood that both antibodies would bind to the same off-target ...to - particularly when you start using complex samples such as whole cell lysate or tissue sections. It...require variable expression of your target across samples - it is hard to distinguish between specific, invariable...there is a wealth of transcriptomic data from this sample type, so you use your bioinformatics skills (or...

...SnapGene Viewer (Mac/PC) FinchTV(Mac/PC) Chromas (PC) 2. You should see individual, sharp and evenly spaced...less and you might suspect contamination in your sample or consider asking your sequencing facility to ...Use a silica spin column for purification of the samples you send for DNA sequencing If your sequencing ...

... fluorescent proteins expressed in the specimen.  2. Fluorescence Activated Cell Sorter (FACs) A machine...photostability of your fluorescent protein when using 2-photon microscopy as this hasn't been well reported...PMID: 18703742. PubMed Central PMCID: PMC2606057. 2. Chtanova, Tatyana, et al. "Dynamics of neutrophil...depth. Two low-energy photons (typically from the same laser) cooperate to cause a higher-energy electronic...

...Transgenesis." PLoS ONE. 8(12): e83043 (20 December 2013). 2. Carola Engler et al. "A One Pot, One Step, Precision...toolkit - aptly named GreenGate – offers all of those same advantages to researchers working in plant model...allows us to design modules that always have the same recognition site but distinct sites where the enzyme...

...linear region to solve for total expression time. 2. Timer functionality with a single gene Although it...290.5496 (2000): 1585-1588. PubMed PMID: 11090358. 2. Tsuboi, Takashi, et al. "Age-dependent preferential...and look at them under a microscope, fractionate samples to isolate particular organelles and their contents...

...;550(1-2):251-268. doi: 10.1016/j.ijpharm.2018.08.043. Epub 2018 Aug 23. PMID: 30145245. Samiul Amin, ...and particle formation. J Pharm Sci. 2015 Feb;104(2):602-11. doi: 10.1002/jps.24259. Epub 2014 Nov 21....

...of these neurons via the channel Channelrhodopsin 2 (ChR2) reduced glutamate transmission from the mPFC...deliver Cre-dependent expression of  Channelrhodopsin 2 (ChR2), a modified receptor protein that activates...actively swimming versus passive floating. In the same paper, the authors used lentivirus to introduce ...

...selection. Desfarges S, Ciuffi A. Viruses. 2010. Jan;2(1):111-30. PubMed . Altering the tropism of lentiviral...Nerhbass U, Montagnier L, Charneau P. Cell. 2000. 101(2): 173-185. PubMed . Woodchuck Hepatitis Virus Posttranscriptional...Lentiviruses can be used to make stable cell lines in the same manner as standard retroviruses. That is, many lentiviral...