...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...

...recombinases like Cre or Flp in their cells of interest and recombinase-dependent AAV vectors for selective...to achieve adequate expression in your cells of interest.  The AAV serotype used can exhibit profound differences...you are using transduces cells that you are not interested in with greater efficiency, it could lead to ...understand which serotype best transduces your cells of interest. We recommend testing several different serotypes...determine which one is the best for your cells of interest.  Determine injection titers and volumes based...injection parameters needed to transduce your cells of interest. Using a constitutively expressing vector for ...is important to test it in the brain region of interest, especially when using DIO/FLEx vectors for off-target...

...precise location of the protein or structure of interest. Endogenous gene tagging is an ideal solution ...creating the plasmids was to identify the gene of interest and decide which terminus, N or C, to tag and ...researchers who study the protein or structure of interest. For a C-terminal tag, as shown in the example...contains 1 kb homology arms matching the gene of interest, a linker, and the GFP tag sequence to be inserted...near either the beginning or end of the gene of interest —in our case in the genome of an hiPS cell. While...this example, at the C-terminus of the gene of interest. To get the molecular components of CRISPR/Cas9...plasmids for introducing tags into your gene of interest. Researchers have developed a variety of endogenous...

...antibody that is specific to the protein you are interested in detecting in your sample. When an ELISA kit...wells, and the antibodies bind to your protein of interest.  The next step is to detect the protein-antibody... sample. However, in this case your protein of interest is pre-incubated with a known amount of an antibody...antibody (meaning it hasn’t bound to your protein of interest) will bind to the competing antigen in the wells...inverse measure of the amount of your protein of interest in the original sample. The competitive ELISA ...sandwich or competitive ELISA because the protein of interest is then detected by an enzyme-conjugated antibody...used to calculate the amount of your protein of interest that is present. Using a Standard Curve To do ...

...impact the antibody’s binding to your protein of interest. Protein preparation Before you can blot your ...incubation temperature for your specific protein(s) of interest.  Determining linear detection range If you are...depends mostly on the weight of the proteins you’re interested in. Table 1 lists the most common types of gels...in a variety of pore sizes. If your protein of interest is small, you may want to consider a smaller pore... size (0.2 µm). Conversely, if your protein of interest is large, look for a larger pore size (0.45 µm... and are worth considering if your proteins of interest vary wildly in size. However, these systems are...It’s fairly easy to decide!  If your protein of interest is available in pure form, you can use it to create...

...University of Amsterdam). Tagging a protein of interest with a fluorescent protein to study its function...all of the native properties of the protein of interest can be challenging. In this blog I discuss strategies...relatively long linkers between the protein of interest and the fluorescent protein. These were either...fusions at the N- and C- termini of your protein of interest The most straightforward fusion protein is made...C-terminus of the fluorescent protein and protein of interest (POI). To indicate the sequence, people often ...mScarlet-tubulin (Plasmid #85045). We often use restriction enzymes in the multiple cloning site for generating...Inserting a fluorescent protein within your protein of interest We have encountered several proteins for which...

...ve gotten the plasmid encoding your protein of interest from Addgene, transfected it into your target ...lane can help confirm the size of the protein of interest. An electric current applied to the gel causes...with a primary antibody against your protein of interest. But which antibody should you use? You will likely...different antibody options targeting your protein of interest from a variety of vendors and you may even find...expression knocked down or knocked out. If you are interested in making your own knockout line for validation... Bauer's labs.    Visualizing your protein of interest with the help of secondary antibodies In order...the primary antibody, which binds the protein of interest, is not labeled. Instead, a conjugated species-specific...

...more easily. Attend special interest group sessions that match your interests. I favor small conferences...provide the opportunity to meet people who are interested in the same things you are on a deep level. Right...common. Namely, the scientific question you are interested in and this is a great ice breaker. Of course...relationships go further and grow over time, but being interested in the same phosphate of your favorite kinase...natural time to meet someone since you are both interested in the same talk. Read more on this advice to...say so. If you read a paper that you think would interest them, send it and ask for their input. Connect...

...gene of interest (or genes) need to be 4.7 kb or less to fit in a single rAAV. For those interested in heftier...icosahedral capsid, that can be used to express genes of interest in cell and animal models. AAVs are non-pathogenic...host cell. In order to make room for a gene of interest, AAVs used in research have their genomes “gutted... than in vitro. On the other hand, if you’re interested in expressing multiple small genes, you can design...expression via switches placed in your cell type of interest. Careful combination of promoters, gene switches...legend from Okada, 2013.   scAAV If your gene of interest is truly small (<2.5Kb) and you need expression...

...technologies and spaces where interested parties can do research. This interest manifested itself in the creation...Over the decades science has become increasingly restricted to academic and industrial labs, but recently...for our computer scientists, many of whom are interested in biology but don’t really know how to start...pipeline. It’s possible that we’ll find something interesting and contribute it back to the kittybiome project...website. Markerspaces differ depending on the interests of their community, and biology is not yet common...recognition, he returned to school to merge his interests in biology and computing. In 2009, he founded ...

...in nearly any neuroscience project or topic of interest. Despite its power and versatility, the technique...experiment to test for sufficiency of your circuit of interest in promoting reward is a self-stimulation experiment...implantation of the optical fiber into the region of interest 3) Behavioral experimentation I’ll discuss some...construct in the brain region and cell type of interest is obviously of critical importance to any optogenetic...promoters and other genetic manipulations to help restrict expression to a specific neuronal subtype. However...constructs are injected directly into the region of interest using stereotaxic surgery (stereotaxic surgery...the proper coordinates of your brain region of interest (as shown in the figure below). Since most of ...

...hierarchical approaches using different Type IIS restriction enzyme specificities at each step. Factors such...limitations through the use of engineered Type IIS restriction enzymes and careful choice of junction sequences...involves the simultaneous activities of a Type IIS restriction enzyme such as BsaI, and a DNA ligase such as...receives the assembly after similar Type IIS restriction enzyme cutting results in 5’ four base overhangs... provide optimal reaction temps for both the restriction enzyme and ligase, thereby enhancing the ordered...destination construct and functioning Type IIS restriction enzyme and DNA ligase, any single omission should...re-engineering of the original BsaI-HF® Type IIS restriction enzyme and the completion of T4 DNA Ligase fidelity...

... Perhaps interest has simply waned. When attendance slumps below a certain level or interest wanes off...things to think about. Choosing a topic and gauging interest The first thing to do if you’ve thought about ...club is to find a topic and seeing if others are interested. You don’t want to pick a topic and have no one...directly involved in the project.  Once we gauged interest from relevant parties, and the company at large... stay caught up and to allow those who may be interested in attending to see what they are missing!  So...institutional inertia drives the meeting rather than interest. These are the journal clubs where participants...

...presence and quantity of DNA, RNA or a mutation of interest. Together SHERLOCK and DETECTR demonstrate the...be programmed with crRNA to target an ssRNA of interest, for example, a sequence specific to a virus or...Csm6 can cleave ssRNA complementary to a crRNA of interest (Niewoehner and Jinek, 2016) and is conveniently..., binding of Cas13 to the programmed region of interest would lead to both the cleavage of a Cas13 specific...flow strips, the presence of your DNA or RNA of interest within a given sample is simply determined by ... When Cas12a-cRNA base pairs with the dsDNA of interest, the DNase activity of Cas12a is initiated. Surrounding... signal designates the presence of your DNA of interest, in this case HPV. As proof of concept, the lab...

...you do will be determined by the topic you’re interested in studying, but, in today’s post, we’ll discuss...short blog post, but some examples I’m personally interested in include: intracranial self-stimulation (ICSS...neurobiology of addictive diseases, which means we are interested in circuits that mediate drug taking behavior...basal neuronal activity in your brain region of interest could be low or highly variable without the behavioral...around a behavioral test in which your circuit of interest is putatively involved in regulating the behavioral...behavioral output. For example, if you are interested in studying a brain region that may be important in...Jeanne Kreek at the Rockefeller University.  His interests include the role of learning and memory in opioid...

...is often our first clue about where the gene of interest might be located. By easily using BLAST to check...additional verification experiments: Primer and restriction digest design Some regions of plasmids are difficult...present in the plasmid, we might need to perform a restriction digest to confirm the size of the plasmid and...including our most popular primers and certain restriction enzymes, but you will also be able to create ...annotations and design additional experiments like restriction digests. We’ll have a tutorial showing all of...Basic Molecular Biology Protocols Diagnostic Restriction Digest Protocol ...

...different plasmids containing the same protein of interest tagged with different fusion proteins for different...dye or other functional ligand, your protein of interest will end up linked to whatever molecule you choose...useful ligand, gray structure is the protein of interest, and black is the fusion tag or labeling system...attach a ligand to a peptide tag on your protein of interest or using non-natural amino acids to directly incorporate...minimally-disruptive genetic tag on the protein of interest. Table 1: Summary of popular self-labeling tag...orthogonal self-labeling tags on different proteins of interest (or one self-labeling tag and a fluorescent protein...sample to label sub-populations of your protein of interest. For example, the Cohen lab used HaloTag for their...

...I would be interested in joining the audit committee about five years ago. I was interested in what she...there will always be an interest in sharing, there will always be an interest in protecting intellectual...the open science movement is that it provides interesting and unique challenges for intellectual property...that balance is, and will continue to be, an interesting legal and professional challenge.Paratore: The...

...depending on your interests. You can look at something as basic as survival. For those interested in development...straightforward. Basically, you place your gene of interest in a plasmid and get the vector incorporated into...transgene. One such marker is the white gene, which restores eye color to fly lines where the endogenous white...fly and downstream experiments Once your gene of interest is cloned into a plasmid, you can send your plasmid...development, organs of interest, such as the intestines, can be dissected at various times throughout the lifespan...