...modifications exist in the template DNA. There are several unique HDR mechanisms, but all templated-repair pathways...fix a broken relationship. Pardo B, et al. Cell Mol Life Sci (2009). PubMed. Regulation of DNA double-strand...along the way. HDR uses homologous sequence as a template for repair and is thus the more accurate mechanism...process is generally error-free provided the DNA template used for repair is identical to the original DNA...one strand of the homologous DNA donor and can template repair off the other strand; this results in the...mechanism that notably does not require a donor template and thus won’t be covered in detail here. Classical...pathway, the 3’ ends invade an intact homologous template to serve as a primer for DNA repair synthesis,...

... region that has mono-methylated and acetylated histones, but not tri-methylated histones, functions as...denote enhancers as follows: (mono_methylation + acetylation) - tri_methylation In the next section we’ll ... me3, and ac – denoting mono-methylation, tri-methylation, and aceylation, respectively (these are chemical...sequencing to measure the relative proportion of guide RNAs (gRNAs) across the population. Recall that guide ...inclusive markers of regulatory regions in the genome, including enhancers, promoters, insulators, and more. I...It Locates ChIP-seq Proteins (for our example later, histones) ChiRP-seq RNA Hi-C DNA (genome-to-genome...tried and true method of finding enhancers by isolating genomic regions that are bound to modified proteins...

...gene-function relationships in defined substructures of the CNS as well as individual neuronal populations.  The...some unknown regulators of neuronal differentiation were enriched in the CD8+ population, as expected....neurons, forming the neuronal circuits that will later be refined through experience. However, studying...complicated by the lack of precise genetic tools for modulating gene expression in the central nervous system...using CRISPRa to target individual genes can be relatively laborious because multiple plasmids/viruses are...generated from the same gene but with different untranslated or coding regions. Typically, each gene isoform...can be targeted anywhere in the genome, it is relatively straightforward to use CRISPRa to selectively...

...know if you are actually stimulating or inhibiting the targeted neuronal population prior to behavioral testing...receive different types of stimulation may help to refine and optimize your stimulation parameters as well. ... and stimulate them for 30min to an hour and observe what happens. You never know how stimulation of a...personally interested in include: intracranial self-stimulation (ICSS) and place preference. The lab I work in... electrode and non-specifically electrically stimulating an ill-defined region of the brain. With the ...particular hole (the active hole) induces laser stimulation of the chosen brain circuit, while a nose-poke...different patterns and the animal only receives stimulation in one of the two chambers (see image below)....

...the model organisms which I use to study gene regulation during developmental processes, CRISPR/Cas9 allows...but currently requires additional experimental manipulation. In some cases, injection of Cre is required...mutation must be removed, which can be done by isolating animals with particular visible phenotypes. These...following a report that one can use linear repair templates (ie. PCR-derived dsDNA) with 30-60 basepair homology...ribonucleoprotein complex with linear DNA as repair templates which reduces time to generate edits to only four...but require variable amounts of experimental manipulation and screening.  A co-selection strategy for ...be obtained in eight days. The only animals on a plate, other than the parental animal, are rescued progeny...

...developing a general translation initiation sequence known as the Species-Independent Translational Sequence (SITS... fuse custom PUF proteins to a translational repressor to down-regulate a gene of interest. You can generate...: 1147. Cooke, Amy, et al. "Targeted translational regulation using the PUF protein family scaffold....extensive sequences used for cloning into the final translated products. Negative selection markers built into...Tikhonova, Elena B., et al. "A transcription blocker isolated from a designed repeat protein combinatorial library...of which you can clone your gene of interest. Translation of the standardized leader peptide prevents secondary...encoding your gene of interest from affecting its translation initiation and instead makes initiation heavily...

...Forward and reverse screens involve manipulating or mutagenizing a population (typically cells or small organisms...screens, there is random or unknown manipulation of the population. This is contrary to reverse screens...disease as the input population. For these experiments, the goal is to identify manipulations which either enhance...in that only survivable manipulations are assessed, rather than all manipulation events. To put it another...looking at cells that can survive. Instead, all manipulations are assessed, regardless of whether they enhance... is assessed. The two most common methods of manipulation are genetic knockout mutations and treatment...consider these questions to save yourself a headache later on. Enhancer and suppressor screens These types ...

...rate of translation and thus protein expression. In fact studies have shown that translational efficiency...particular organism. Why is codon usage important? Translation and protein synthesis relies on transfer RNAs...efficiency is correlated with codon bias across all endogenous genes in E. coli and S. cerevisiae (Tuller et.... Image from Mariana Ruiz Villarreal. By the late 1980s, scientists even created a codon adaptation... 2000 the Ikemura lab created the Codon Usage Tabulated from GenBank database (CUTG), that provides an...on an organisms codon usage bias to increase translational efficiency and thus protein expression without...an organism that contains expression limiting regulatory elements or use a non-canonical code (Gustafsson...

...the resistant populations. In fact, 88% of the barcodes within the resistant populations were shared by...screening with lentiviral pooled shRNA libraries. Later, Bhang says, “When we developed this tool, there...library to determine whether or not cancer cell populations resistant to drug treatments arise during treatment...treatment or are preexisting in the parent population prior to the start of treatment. They hypothesized...hypothesized that, if they labeled the parent cell population using the library, propagated it, separated it into...cells that represent the preexisting resistant subpopulation 2. Resistant cells among the separate replicates...that are present at less than 0.1% of the total population are easily be missed. The ClonTracer library ...

...ratio calculators Lab math calculators These calculators can help you with common calculations performed...lab. Molarity calculator Promega's Biomath Calculators Primer resuspension calculator (sign-in required...sequences to sequences in a database ExPASy translation tool: Translates a DNA sequence using all six open reading... Cloning ligation reaction calculators These links can help you calculate how much insert and backbone...Annotates features, BLASTs, aligns, digests, and translates a sequence.   NEBCutter2: For finding restriction...information, such as expression patterns, post-translational modifications and polymorphisms Codon optimization...Addgene's Analyze Sequence tool.   DNA sequence manipulation tools  Sometimes DNA sequences need to be “cleaned...

... mk+) Unmethylated DNA not degraded, cell still can methylate DNA   hsdS(rk-,mk-) Unmethylated DNA not...from two individual isolates, the K-12 strain and the B strain. K-12 was isolated from a patient in 1920...pathway that cleaves methylated cytosine DNA Allows for uptake of foreign (methylated) DNA mrr, Δ(mcrC-mrr...that cleaves methylated adenine or cytosine DNA Allows for uptake of foreign (methylated) DNA recA, ...plasmids that should not be Dam or Dcm methylated, allows for methylation sensitive restriction enzymes to ...renaming samples throughout history. It was likely isolated in 1918 but was first referred to as “B strain...routine cloning, cloning unstable DNA, preparing unmethylated DNA, and more. Many mutations that make these...

...strands of the template DNA first have to be separated. This occurs by heating the dsDNA template to a point...categories of multiplex PCR:  Single template PCR reaction - one template is amplified using several forward... reverse primer sets.  Multiple template PCR - multiple templates with different primer pairs that align...invented in the early 1980s by Kary B. Mullis who later shared a Nobel Prize in Chemistry for his work. ... in temperature. Taq was first identified in the late 1960s during research at hot springs in Yellowstone...polymerase, PCR requires free nucleotides (dNTPs), template DNA to amplify from and unique single stranded... primers can anneal to the single stranded DNA template strands. DNA polymerase is also stable at this...

... Single-stranded DNA repair templates Short single-stranded DNA templates have been shown to improve the...final ssDNA template. Figure 2: Overview of ivTRT to generate an ssDNA repair template. Although...laboratory mouse. It’s also made targeted insertions relatively simple in C. elegans and bacteria. But CRISPRing...from low efficiency when a standard dsDNA repair template is used. The original design for creating a conditional...targeting two intronic regions and two repair templates in order to insert two loxP sites. Since two double-strand...ameliorate this problem. Briefly, you design your donor template to include a T7 promoter upstream of the left ...vitro transcription, and use the IVT product as a template for reverse transcription to create your ssDNA...

...As with DNA isolation, scientists commonly rely on RNA isolation kits to make their life easier. Recently... out. In this article, we cover the basics of isolating RNA without a kit. Tips for working with RNA (... today There are many alternative methods for isolating DNA without a kit. However, that isn’t the case...method. A major hurdle to developing protocols to isolate RNA was that RNases are commonly found in cells...upon cell lysis, RNA is degraded. To effectively isolate intact RNA, a fast, strong protein denaturant would...chance to break down RNA upon cell lysis.  In the late 1970’s, Chirgwin and colleagues showed that a strong...., 1979). They developed a protocol meant for isolating RNA from rat spleens in which they homogenized...

...detect proteins within a 96- or 384-well, flat-bottomed plate. The basic principle of an ELISA is that ...technique works by first coating the wells of a flat bottomed plate with a capture antibody that is specific ...experimental sample are bound to the wells of a plate instead of an antibody. Coating the wells with a...characterize antibodies against the antigen bound to the plate. Figure 3: Overview of using Direct ELISA...protein antigen is coated onto the wells of the plate. However, instead of detecting your protein using...absorbance of each well, which is measured using a microplate reader, is directly proportional to the amount... of enzyme present in the well and is used to calculate the amount of your protein of interest that is...

...formation of disulfide bridges, glycosylation and acetylation to create functional, stable proteins. Protein...used to manipulate DNA), or in medicine (e.g. insulin). Proteins, unlike DNA which can be relatively easily...cells with your DNA template of choice and allowing these cells to transcribe, translate and modify your ...transcribed into messenger RNA (mRNA). mRNA can then be translated at the ribosome into polypeptide chains that ...proteins are then modified via an array of post-translational modifications including protein folding, formation...proteins from mRNA and the addition of post-translational modifications Learn more molecular biology ...mammalian proteins that require multiple post-translation modifications for proper protein function. Most...

... the biotinalatable tag on dCas9 with a BioTAP-tag. The BioTAP-tag is a 69 aa long biotinylation targeting...Jul 2, 2020. Promoters may be the star of gene regulation, but enhancers and chromatin looping play important...important supporting roles. Enhancers are cis regulatory DNA sequences that, when bound by transcription...thousands of base pairs away from the gene they regulate, but are brought in proximity by the looping of...CAPTURE (CRISPR Affinity Purification in situ of Regulatory Elements) method. The original CAPTURE method...2020). CRISPR Affinity Purification in situ of Regulatory Elements: The original CAPTURE method The original...the dCas9 to the loci of interest and dCas9 is biotinylated by BirA. From here, the CAPTURE method is similar...

...antibody manipulation), then expression is likely sufficient for successful optical stimulation.  It is...Gore BB, et al. Manipulating gene expression in projection-specific neuronal populations using combinatorial...technique is remarkably straightforward to learn and relatively cheap (compared to many other technologies and...electrophysiology to prove that your optogenetic manipulations are generating action potentials (or lack there...assess the effectiveness of your optogenetic manipulations (though a reviewer will probably ask for electrophysiology...circuit of interest in promoting reward is a self-stimulation experiment wherein mouse initiated optogenetic...in a reward sensation and subsequent repeated stimulation. A spatial memory task, on the other hand, would...

...possible to build gene regulatory RSMs (GRSMs, the subset of RSMs that regulate gene expression) that ...massive amount of information regarding which regulatory proteins, signaling molecules, mutations, and...individual bacterial cells to form colonies on a plate and then we performed colony PCR on the register...sequencing.  A less straightforward, but more population-wide (as opposed to single-cell) approach is ...a particular application makes it difficult to isolate single registers or if it requires reading so many...necessarily have to be based on transcriptional regulation, as long as they can control recombinase activity...post-transcriptional recombinase activation via RNA toehold riboregulation could be a promising strategy for linking RSM...

...Kinases: they regulate many proteins, with ~1/3 of human proteins predicted to be phosphorylated on at least...least one site. Phosphorylation is particularly important for regulating signal transduction and measuring...predict. Slower Dephosphorylation Rates: FRET-based reporters can have slower dephosphorylation rates than ... signal (NES), both which are phosphorylatable. Prior to phosphorylation, the KTR localizes to the nucleus... of the nucleus depending on its phosphorylation status. The relative ratio of cytoplasmic vs. nuclear...hypothesized to be caused by FRET reporters having phosphorylated conformations that are less phosphatase-accessible...nucleus, but following phosphorylation bNLS activity is suppressed and NES activity is enhanced, leading...