...cognate peptide epitopes fused to the protein of interest are recognized and thus fluorescently labeled ...modified to use different antibiotic resistances, restriction enzymes and plasmid backbones. pUCXKT contains...fusion of the antibiotic cassette to the gene of interest. The gene to be expressed is amplified using a...gene-specific forward primer containing the desired restriction site (the MCS has several options) and a pUCX-specific...resistance cassette, a ribosome binding site, SacI restriction site and a linker region. The PCR product is ...used with other entry clones encoding the ORFs of interest: Buj et al., BMC Molecular Biology. 2013 August...A desired insert is cloned using traditional restriction enzyme cloning to create an Entry vector containing...

...pair of restriction enzymes. These enzymes, Nt.BbvCI and Nb.BbvCI, recognize the same restriction site, ...multiple amino acid changes affect a protein of interest’s function. This technique relies on the generation...deep mutational scanning.     3 reasons to be interested in deep mutational scanning Hundreds of thousands...variable bases (NNN) at any given codon site of interest can be cloned into an expression vector with Gibson...mutagenic primers that tile across a region of interest, generating multiple mutants in one PCR reaction...primer sets tile across the protein or region of interest. A low primer-to-template ratio is used to promote... be sense. Does the orientation of the BbcVI restrict site matter? Most of the time no, but there are...

...information in the app is restricted to the NEB inventory only, the restriction enzyme sequence resource...smartphone nearby. We tailor these devices to our own interests with the content we store in them: music, emails...comes in handy when I use protocols that require restriction enzymes or PCR. The app contains a double digest...digest tool that is only helpful for NEB restriction enzymes since the instructions use NEB buffers. Nonetheless...name to discover all the information about that restriction enzyme or share it via email with a colleague...colleague. The app is also great if you’re adding a restriction site to a primer, you only need to skim the alphabet...screen to find the sequence for your enzyme of interest. Lastly, the Tm calculator helps you set up an...

...TANGO) (Kroeze et al., 2015). PRESTO-TANGO uses a modified Tango beta-arrestin recruitment assay to measure... examples with plasmids deposited at Addgene.  PRESTO-TANGO: The ligands that activate many GPCRs are...Roth’s lab created an open source resource called PRESTO-TANGO (parallel receptorome expression and screening...this assay, binding of an agonist recruits a beta-arrestin-TEV protease fusion to a receptor-transcription...ligands for unknown GPCRs. You can find the entire PRESTO-TANGO kit at Addgene! TRUPATH: Bryan Roth’s lab... G proteins activated by ligand-bound GPCRs of interest. The TRUPATH plasmid kit consists of 20 plasmids...for more information on this system! GPCR-APEX: Interested in tracking the downstream signaling pathways...

...use restriction digest to screen your clones, but there are many other ways to choose restriction enzymes...Blue-white screens rely on vectors where the gene of interest is inserted into the middle of the lacZ gene, ...blue-white screen. Image from Stefan Walkowski.   Restriction digest Another way to distinguish between colonies...inserts from those that do not is by using a restriction digest. The key is to be strategic about your...on whether the plasmid contains your insert of interest or not. For example, choose enzymes that only ...route, check out our protocol video on using restriction digests to analyze plasmids, which can help you... you visualize this process, or visit our restriction analysis blog post. Colony PCR Colony PCR can...

...If you study proteins, you’re probably quite interested in the canonical 20 amino acids. But in your ...your quest to learn more about your protein of interest, you may find the available amino acids a bit…limiting...Shandell et al., 2021).   How it works If you’re interested in using GCE, here are the basic requirements...plasmid will be needed to introduce the gene of interest containing the chosen codon that will result in...of the non-canonical amino acid at the site of interest (figure 1). While the codon used is often one ...getting high enough expression of your protein of interest. If that wasn’t enough, in this development-focused...introducing ncAAs with protein modifications of interest or any of a huge variety of ncAAs to study the...

...to you as a researcher. National interest waiver The National Interest Waiver (NIW) allows for self-sponsorship...as the National Interest of the EB-1A. Furthermore, the ability to leverage the prestige of the employer...Cap Subject Industry H1B Academic H1B National Interest Waiver EB-1A EB-1B Cap subject industry H-1B...opportunities to develop skills related to their areas of interest. Additionally, since most universities and non-profit...determine if an applicant qualifies for the National Interest Waiver. The three prongs are: (1) The foreign ... and plan for future activities; including the interest of the greater scientific field, potential customers...must be able to show that you serve the national interest to a substantially greater degree than the majority...

...method to identify the expression of transgenes of interest. Furthermore, fluorophore variants (for example...simultaneous identification of more than one protein of interest. However, in our experience, we found that fluorescent...than an aid, in the expression of the protein of interest. The green fluorescent protein (GFP) and mCherry...interfere with the correct function of the protein of interest. In our studies, we used AAV viral vectors to ...influence on the expression of the transgene of interest and its localization in the membrane. The lesson... can impact the expression of the transgene of interest. In our studies, mCherry interfered with the transport...could help improve the transport of protein of interest the membrane.   Figure 1: Electron microscope...

...-in of fluorescent tags into the gene locus of interest. An advantage of this alternative is the possibility...in situ. I was in luck when I attended a very interesting talk by Prof. Erin M Schuman, a former Caltech...antibodies directed towards two endogenous proteins of interests, suspected to interact, are added to a cellular... strand added to them. Only if the proteins of interest are in close proximity to each other can the DNA... one antibody was directed toward a protein of interest but the second antibody was directed toward AHA...antibodies recognized an endogenous protein of interest that also had incorporated the AHA tag after pulse...time. Thus, only newly synthesized proteins of interest were detected and later visualized by FUNCAT-PLA...

...including more background on how your virus of interest causes cancer, as well as therapeutic strategies...the conference, map out which posters you are interested in viewing. For most larger conferences, poster...session, so bookmark which posters are of most interest to you a few days in advance. If you don’t have...If you come prepared with a list of posters of interest, then you’ve already set yourself up for success... – kindly let the presenter know you are very interested in his/her work, and would enjoy hearing the ...constraints, there may be particular poster you are interested in viewing which has drawn a large crowd, making...Time permitting, you can go back to the most interesting and popular posters at the end of the session...

... do during your training is meet interesting people doing interesting things. I call this building relationships...learning new things from other scientists doing interesting work. Consider this to be like any other research...research project. You’ve met someone whose career interests you or you want to pursue someone doing a job ...nonthreatening request to discuss experience, interests and career path. Because there is no demand for...what networking is about), but be upfront about interest in a job. Ask openly to be connected to someone...people like to help One good place to look for interesting connections is your university’s alumni database...too busy to respond to you). You will uncover interesting opportunities by meeting with people working ...

...regions. If you’re interested in studying the brain in a way that goes beyond such restrictions, you may want...Mich et al., 2021) can help restrict expression to your cell type of interest, while miRNA target sites ...large or diffuse biological structures. Of primary interest here are systemic capsids that target the central..., including species, strain, age, sex, area of interest, and health. While it’s always helpful to start...your average AAVs — and have many of the same restrictions, like limited cargo space (~4.7 kb) for your...used to prevent expression in cell types not of interest. Systemic capsids packaging Cre-dependent transgenes... with Cre-expressing transgenic mouse lines to restrict transgene expression to specific tissues and cell...

...Harris Lab relies on the restoration of a frame-shift in mCherry that restores fluorescence to monitor ...that disrupts fluorescence due to a frameshift. Restoration of fluorescence can only occur through editing...results in two DSBs that can be repaired by NHEJ restoring mCherry expression. Cells can then be sorted using...C-to-T editing by the APOBEC-Cas9 editosome would restore eGFP fluorescence, making it easy to quantify single... point mutations in eGFP that results in the restoration of fluorescence. Transient reporter for editing...-GFP conversion and to target specific loci of interest in the genome. The Wang and Brafman lab showed...higher frequency of genomic base pair edits of interest compared to cells segregated solely by a reporter...

...Comparison Between Standard Restriction Cloning and Fast Cloning Requirement Restriction Cloning Fast Cloning...elements of biomedical research. Traditional restriction cloning is one of the oldest of these techniques...purification, ligation, and transformation. While restriction cloning is still routinely performed by many ...Cloning Choose Restriction Enzymes + - PCR Amplification + + Digestion + + Gel Purification + - ...products and digest with methylation sensitive restriction enzyme DpnI to remove the templates. Digestion...graduate student at the University of Connecticut interested in protein science.     References: 1. Celie,... restriction cloning...

...cells of interest do not lie beyond the dimensions displayed in the plot. (B) A more restrictive gate results...of my cells of interest lies within a clear negative population. When you’re interested in measuring a... to select and precisely measure your cells of interest. In many experiments you’ll be working with a ... on the following gates, cells that are not of interest can still be gated out later. Based on personal...(single cells, live cells, my marker stains of interest). Let’s double-click inside our gate and proceed...you have identified and gated on your cells of interest, you will likely need to exclude all duplicate...multiple individual gates around the populations of interest (Figure 6). A.                                                                                               ...

...cloned with an ORF of interest for customized experiments. Once the ORF of interest is cloned into one of...phosphomimetic residues. The phosphorylated residue of interest is mutated to a negatively charged residue, aspartate...plasmid encoding the open reading frame (ORF) of interest (Figure 2). 1. The phosphoserine OTS The backbone...a plasmid encoding their open reading frame of interest. Two expression systems have been verified for...instead of a glutamate which allows the system of interest to be validated first through the production of... generate as much pSer-incorporated protein of interest as possible, some optimization will be necessary...to a single phosphate moiety on the peptide of interest.  After confirming pSer incorporation, researchers...

... and interests. Are you particularly good at writing and editing manuscripts? Are you interested in scientific...science professions based on their skills and interests. Once scientists identify potential career pathways... academic setting and to shape and strengthen interests and skills. Although the word “extracurricular... potential careers that suit these skills and interests: Use career planning tools such as the Individual...opportunities beyond academia. Once a profession sounds interesting, TAKE ACTION! Taking action: Examples of extracurricular...outside your particular field of study Assess your interest in a different field Outreach activities...information about shaping your career to your values, interests, and skills? Curious to know where graduate students...

...unique restriction enzyme site and unique primer sites for more rapid screening by restriction digest ..., et al. Cell Rep. 2015. PubMed PMID: 26190106 PRESTO-TANGO: An open-source resource for drug discovery...ligands for orphan GPCRs is of significant medical interest, however efforts to do so have been hindered by...receptors. Bryan Roth's lab has a newly available PRESTO-TANGO kit which allows researchers to interrogate...publication, Kroeze et al. describe their enhanced TANGO arrestin recruitment assay incorporating 314 codon-optimized...and Screening via Transcriptional Output-TANGO (PRESTO-TANGO) to screen the NCC-1 library of approved ...discovery. Researchers can use plasmids from the PRESTO-TANGO kit directly for drug screening or easily...

...efficiencies that may be low in the cell line of interest. The piggyBac™ system allows for significantly...of the donor-transposon (carrying your cargo of interest) into random ‘TTAA’ sites throughout the genome...line-dependent, and should be tested in the cell line of interest. Importantly, by varying these ratios, you can... or minimize expression levels of your gene of interest, as needed. Controlling the expression levels ...isolating the specific cells that have the edit of interest. Fortunately, by combing nuclease technology with...transposon in a donor plasmid carrying the DNA edit of interest, along with a site-specific nuclease (such as ...thousand clones to find a cell line with the edit of interest. With the larger piggyBac™ donor vector, it is...

...protein, and then voila! Wherever your protein of interest is, a signal will also be. This is the direct ...giving you enough signal to detect your protein of interest, it may be time to look at the indirect approach...While a primary antibody binds to the protein of interest, a secondary antibody binds to the primary antibody...molecules associated, indirectly, with each protein of interest.    Figure 1: The direct approach (left...secondary antibodies are indicated when the protein of interest is found in low amounts or if the conjugated signaling...primary antibody for your assay and protein of interest is often one of the most challenging components...multiplex assay - one with multiple proteins of interest - the secondary antibody needs careful consideration...