...organisms and cell lines. Although ChIP-seq is a relatively simple and robust protocol it does have limitations...study how differential access to transcriptional regulatory elements is controlled during development using...fused micrococcal nuclease (MNase) to specifically isolate DNA that is bound by a protein of interest. First...First, nuclei from tissue or cell culture are isolated using lectin-coated magnetic beads. Nuclei are then...MNase requires for its nuclease activity and is chelated from the reaction up until this point. The nuclease...simple lab protocols Browse CRISPR plasmids for isolating a genomic locus Browse FRET plasmids for studying...

...Stable Cell Lines with Lentivirus Isolating a Monoclonal Cell Population by Limiting Dilution Lentivirus...high Cas9 MOI considering that A549 cells are relatively easy to transduce. I ended up with too many Cas9...be uniform editing across all the cells in the population, so some of the cells will still appear green...efficiently editing, don’t expect a 100% knockout population. BRAF gRNA delivery  Overview: In practice you...on genomic DNA. The surveyor assay itself was relatively easy. The basis of the Surveyor assay is that...get an idea of the percentage of cells in the population that have an edit. I didn’t do this, but to do...

...seeds, DNA and other resources for Arabidopsis and related species. Located at The Ohio State University in...member of the Brassicaceae family, Arabidopsis is related to a variety of food crops such as broccoli, cabbage...utility to science, and its well-known edible relatives, it is fair to say that most people do not recognize...teaching community through the TRAINED initiative (Translating Research on Arabidopsis Into a Network of Educational...through TAIR with a new ABRC database and ordering platform soon to be released. For individuals interested...Arabidopsis is small, has a short life cycle, and is relatively easy to grow, it is an ideal organism for teachers...

...viral preps   Improved cell-free transcription/translation system to examine biosynthetic pathways  By Gabrielle...Gabrielle Clouse Recently, cell-free transcription/translation systems (also referred to as TX-TL systems) have...In summary, this cell free system allows for a relatively simple, controlled “one-pot” reaction capable...plasmids that helps identify base edited cell populations without end-point sequencing assays. These plasmids..., or XMAS-TREE, to identify base edited cell populations without end-point sequencing assays.  This tool...different vectors and flow cytometry is used to sort populations by mCherry and GFP expression to check for ABE...

...balance between positive and negative regulation and can be manipulated with mutations in the replicon. For...produce just a few copies depending on how they are regulated. Generally, control of replication is referred...stringent" depending on whether the ori is positively regulated by RNA or proteins, respectively. A plasmid's ...plasmid copy number  Although the sequence and regulation of the ori dramatically affect the copy number...SB, Furman JL, Xiao H, Schultz PG. A versatile platform for single- and multiple-unnatural amino acid ...

...polyA  The polyadenylation or polyA signal aids in the nuclear export of RNA and RNA translation, and promotes...plasmid. Parts shown in grey are optional cis-regulatory elements. Key elements of an AAV plasmid First...plasmid harbors non-coding sequences. Other cis-regulatory elements Next let’s talk about some of the elements...Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) forms a tertiary structure when...Technologies and Methods for Targeted Neuronal Manipulation. Front Neuroanat 13: . https://doi.org/10.3389...

...DNA, a cDNA library, or by RT-PCR. Isolate the PCR product Isolate your PCR product from the rest of the...function in the same buffer, as this will save time later In our example, we will use EcoRI and NotI to ...TGGCATATCTCGAAGTACTGAGCGGCCGCTAAGCA-3’) into this calculator we get a final Reverse Primer sequence of 5’-...If you are amplifying from a plasmid or simple template, there is very little chance for mis-priming, ...now ready for restriction digestion. As such, the later steps in this process are the same as those discussed...

...of interest as well as sequences necessary to encapsulate that insert into a viral particle (the packaging...lentiviral vector system is the tetracycline (Tet)-regulated system. You can choose either the Tet-off system...rtTA (FUW-M2rtTA). In this kind of approach, a population of cells might express only the tTA or your transgene...proto-oncogenes or oncogenes for instance should be manipulated with care. See the NIH’s biosafety guidelines...viral capsid antigen p24, the FACS dosing method correlates viral titre to the number of cells transduced...order to aid viral binding. This method, called spinoculation, has been found very effective for T cells transduction...Yasutsugu Suzuki and Youichi Suzuki (2011). Gene Regulatable Lentiviral Vector System, Viral Gene Therapy,...

...BG) (Keppler et al., 2003). This engineered hAGT later became known as SNAP-tag. There is also a fast-reacting...demonstrated in 2005 that proteins fused with E. coli dihydrofolate reductase (eDHFR) could be labeled with fluorescent...Although this tight interaction is not covalent, later work showed that introducing a Leu28Cys mutation...SNAP-OMP25 with Atto590-BG (green), imaged with stimulated emission depletion (STED) nanoscopy. Scale bar...different ligands to the same sample to label sub-populations of your protein of interest. For example, the...-molecule tracking of actin filament assembly-regulating proteins in vitro reveals complex events. a) ...Gaska, I., & Shekhar, S. (2023). Multicomponent regulation of actin barbed end assembly by twinfilin, formin...

...Kurt Thorn (UCSF at the time) began an effort to collate some of the current “best” FPs, and I collaborated...using simple web forms) while also maintaining a relatively strict database design (schema).  Registered ...allows for visual comparison of FPs and how they relate to one another. .  In addition, the FPbase Fluorescence...feature filter spectra and collection efficiency calculations.  With ~80 FP spectra (and rapidly growing, ... page with all the information in the database relating to that specific protein (for instance: EGFP)....retrieve all of the information in the database relating to that set of proteins.  For computer-savvy users...

...might not have to do the calculation yourself as many fluorometers will calculate your sample concentration...than 1.5. Then, using the A260 reading, you can calculate the DNA concentration. Generally, A260 of 1.0 ... start by pouring your gel containing a DNA intercalating dye (ex: ethidium bromide) and choosing a DNA...quantify based on band intensities of your DNA relative to the intensity in the ladder. It’s best to compare...quantified over time using a fluorescent dye that intercalates into the DNA such that smaller fragments are...

...technology (Yesbolatova et al., 2019). Find the mAID tagging toolkit at Addgene Yesbolatova et al., Methods...ubiquitin ligase, the Kanemaki lab was able to translate AID to yeast, as well as mammalian and human cell...tagging of proteins with mAID and other tags (Yesbolatova et al., 2019) using CRISPR. This toolkit makes...thought to play a key role in the transcriptional regulation of many genes. A number of groups have designed...synthetic looping factors developed thus far for these latter experiments, such as transcription factors tethered...

...genetic manipulation more hands on While teaching courses about gene therapy and genetic manipulation, I and...on genetic manipulation of human stem cells when students are not even allowed to manipulate prokaryotic...transfer plasmid that could be used to genetically manipulate human stem cells. Then they had 10 minutes to...explain why they selected a certain target cell population. Who is the winner? Ultimately, society should...

...Watch out for methylation issues. Enzymes like XbaI and ClaI are sensitive to methylation and their activity... Congratulations, you have a plasmid expressing your gene of interest (YGOI) and are ready to dive into...the reaction on an agarose gel to determine the relative sizes of the resulting DNA fragments. Restriction...will need to purify your DNA from a dcm or dam methylation-deficient bacterial strain such as JM110 or INV110...enzymes are working. When uncut plasmid DNA is isolated and run on an agarose gel, you are likely to see...

... Sensitivity Cas9 sensitivity is often closely related to specificity. Sensitivity is a measure of the...site. A specificity ratio can be measured by calculating the on-target activity divided by the off-target... SpCas9 described the PAM specificity of Cas9s isolated from different organisms and engineered Cas9 variants...the enzymes showed decreased off-target activity relative to Cas9. Since then, the Joung and Jennifer Doudna...showed decreased or similar off-target activity, relative to eSpCas9 and SpCas9-HF across a number of different...research due to its small size and specificity relative to Cas9. As scientists continue to characterize...

...devices for research. There are a number of new platforms (such as openUC2, which I am a co-founder of),...that make everyday microscopy much easier. On platforms such as Github, users can download the software...example, can also be printed quickly in Madagascar. Platforms that allow for on-going creativity with the construction...organizations and companies to provide problem-solving platforms.     Fig. 3: Different workflows involved...their work on the shoulders of giants. The still relatively young movement of sharing open hardware has a...Prakash, "Squid: Simplifying Quantitative Imaging Platform Development and Deployment." bioRxiv. 2020. Diederich...

...limited to: rapid growth, ease of replica plating and mutant isolation, a well-defined genetic system, and ...and animals. Unlike bacteria, yeast can post-translationally modify proteins yet they still share many of...a previous post, we briefly discussed how the regulation of bacterial ORIs determines plasmid copy number...these vectors can also be controlled if specific regulatable elements are included (reviewed here). Plasmids...complementation is weak. yes   LYS2 L-lysine yes - Grow on plates containing alpha-aminoadipate in the absence of...

...brightness for a few commonly used fluorophores in relation to the laser type. Table adapted from: https:/...are absorbed and then emitted, which creates a relatively stronger — or brighter — signal.   Figure...use brighter fluorophores for markers that are relatively lower in abundance. In contrast, when you expect...the spectra of enhanced GFP (eGFP) and FITC, the latter being a commonly used fluorophore for antibody-...again. In the beginning, we talked about the relationship between a photon's wavelength and energy, how... into different levels of brightness and their relation to the fluorophore's excitation efficiency, and.... His projects include investigating negative regulators of T cells and cytokine signaling in tumor immunity...

...specific, individual proteins under endogenous regulatory control. But even with the advent of CRISPR/Cas9...the gene to ensure it would be transcribed and translated. For an N-terminally tagged protein, we used ...human genes and will add more to the collection later this year. Designing the donor plasmid A key feature... one thousand base pairs on each side, in fact. Later, when we transfect the cells, these large regions...percent of cases, the stem cell will seek out a template for the repair process. By flooding the cell with...using the introduced donor plasmid as a repair template. This results in the insertion of the GFP tag ...

...purification. In this strategy a single protein can be isolated from a complex mix using affinity chromatography...variety of methods can be used to successfully isolate proteins, this blog will focus on one of the most...the protein of interest and it is successfully isolated.   Prior to the polyhistidine tag, alternative...binding of untagged proteins. Despite being a relatively low frequency amino acid, proteins containing...questions about polyhistidine tags. For more antibody-related information, sign up to receive alerts for antibody...Purification of Recombinant Proteins with a Novel Metal Chelate Adsorbent. Bio/Technology. 1988; 6: 1321–1325. ...