...transform S. cerevisiae met deletion strains with overexpression plasmids carrying MET/Met genes from either...to grow in the absence of methionine. Since the GAL1 promoter controls MET/Met gene expression, complementation... insert the coding sequences for ten different MET/Met genes (Figure 1) from the two yeast species into...without uracil supplementation (6). Expression of MET/Met genes from the pYES2.1 plasmid is controlled by... replica plating to determine if expression of MET/Met genes from the plasmids allows the transformed ... but not all, S. pombe Met genes complement the corresponding S. cerevisiae met deficiencies (manuscript...plasmid-encoded MET gene allows the transformed strain to grow in the absence of methionine (i.e. the plasmid...

...decide which method to use. This post provides a brief overview of reprogramming methods with the goal...Non-integrating methods have a smaller genetic footprint compared to integrating approaches. These methods eliminate...universal method that can handle all applications of iPSCs, but there should be at least one method that can...Mahendra S. Rao. "A Review of the Methods for Human iPSC Derivation." Methods in Molecular Biology Pluripotent...Find Plasmids for Stem Cell Research Delivery Method Mechanism Efficiency Safety Pros Cons MMLV-derived...proteins. Table 1: Key Features of Different Methods for Generating iPSCs. One important note: in this...number maybe misleading. Integrating iPSC delivery methods Overall, integrating viral vectors efficiently ...

...faster to perform. Introduction to flow cytometry Flow cytometry allows users to analyze single cells in...Antibodies-based flow cytometry can use both direct and indirect staining methods. In direct staining, ... will no longer recognize them. Sometimes, users will combine methods and fix with formaldehyde first ...the desired parameter. A droplet containing a cell that is positive for a desired parameter is collected... cultures.  Interpreting flow cytometry data To interpret flow cytometry data users will typically start...   Tips for flow cytometry success Break up cell clumps Success in a flow cytometry experiment depends...protocols? If this sounds like you, then flow cytometry can help you overcome these challenges and make...

... Dcm methylase adds a methyl group to the second cytosine of CCWGG EcoKI methylase adds a methyl group...contain 3 methylases that recognize and methylate different stretches of DNA: Dam methylase adds a methyl...information. Enzyme Dam methylation Dcm methylation EcoKI methylation ApaI Not affected Blocked ...questions is the same--methylation! Read on to learn about how DNA methylation may affect your restriction...The other component of these systems, methyltransferases, methylate DNA at particular sequences to prevent...restriction modification systems containing methyltransferases that add methyl groups to specific DNA sequences ... expression. Methylases involved with these processes (for example Dam and Dcm methylases) are independent...

...Protocols on Addgene's Protocol Pages Agarose cloning method procedure The procedure (illustrated in the YouTube... each well. Tips for using the agarose cloning method Test your agarose temperature. Cloning cells using...well-spaced clones. For rapidly growing cells we sometimes use TPP peelaway T-75 flasks to provide a greater... in outgrowth. Depending on the cell type, we sometimes add a drop of serum to each well in the 48-well...of interest. Advantages of the agarose cloning method The agarose technique should be useful to scientists...experience, cell clones picked using the agarose method do not differ grossly from those isolated using... agarose method...

...When using flow cytometry to analyze your samples, it is necessary to set up a sequence of gates to be...marker expression, and viability. Gates are the parameters the machine uses to differentiate between variations...bit of theoretical background on the different parameters that your gating strategy will be based upon....digitally extract your cells based on their size, the cytometer provides you with two measurements: forward scatter...scatter (SSC). To acquire these measurements, the cytometer illuminates the passing cell with a laser and ...of FSC, SSC, and all fluorescent stains. The cytometer's software will allow you to adjust this setting... and Area Within FSC and SSC, there are three parameters that are being measured: height (H), area (A)...

...conventional flow cytometer.   Spectral flow cytometry Full spectrum flow cytometry, or “spectral flow...panel! Although conventional flow cytometry and spectral flow cytometry are more alike than they are different...which spectral flow cytometry requires special considerations. Conventional flow cytometry During conventional...spectral flow cytometry for a given experiment, it is important to consider how many parameters you want to...Flow cytometry is one of the most powerful tools available to immunologists, allowing for the rapid analysis...such as PBMCs or tumors, and moreso than other methods, the identification and isolation of rare cell ...cell types. In recent years, full spectrum flow cytometry (“spectral flow”) has emerged as a particularly...

...Relationships need nurturing. Perhaps you’ve met someone interesting at an event. Or, perhaps you have...and always include a personal note about where you met and what you discussed. Do this in a timely manner...there’s good news.  Send an email if you have something to ask or say. It can be as simple as “it was ...ask me about the different arms of this starfish sometime, but as of yesterday, I have 4,700 LinkedIn connections...

... K. (2018). Optogenetics and Chemogenetics. In Methods in Enzymology (pp. 181–196). https://doi.org/10.1016...

...complicated, often costly in vitro assembly methods. A new method, REPLACR-mutagenesis, harnesses the power.... Current site directed mutagenesis methods Current SDM methods rely on in vitro assembly. To make a ... DNA is digested with methylated DNA-specific enzyme DpnI, and the nonmethylated PCR products are then...cost. Read on to find out how to replace your SDM method with REPLACR (Recombineering of Ends of Linearized...transformed into E. coli to undergo nick repair. This method can work well, but poor template digestion can ...used to assemble multiple fragments, but these methods can be expensive because they require particular... enzymes, some of which are proprietary. These methods also rely on ssDNA ends to match up regions of ...

...several different methods, so we've summarized some key features of common  delivery methods below: Chemical...These tables are not inclusive of all methods, nor are these methods limited exclusively to in vitro cell... applications for each, and potential delivery methods.   General considerations for planning your CRISPR...Choose an appropriate expression system and delivery method: Do you need stable integration or is transient... make sure your expression system and delivery methods are compatible with all your components. Next, ...expression system, you can then choose the best method for introducing those CRISPR components into your...dividing and non-dividing cells. AAV is least toxic method for in vivo viral delivery. Cas9 mRNA and gRNA...

...gene control. However, these approaches are often met with the challenges of regulatory bottlenecks and...successes in metabolic engineering have been limited to the overexpression of various metabolites in Escherichia...species or metabolic pathways. A new study takes us one step closer to the vision of metabolic biofactories...cellular behaviors, such as differentiation or metabolism. Our attempts to manipulate these cellular behaviors...be host-independent, beyond the oft published metabolic engineering realm of S. cerevisiae and E. coli...control via scRNA design is readily apparent in metabolic engineering. The authors elegantly demonstrated...demonstrated the potential of this to control a metabolic network in S. cerevisiae by using three RNA programs ...

...Many existing methods for identifying PPIs, such as affinity purification mass spectrometry (AP-MS), lack...additional interacting factors. Problems with Existing Methods for Studying Protein-Protein Interactions Difficult... No spatiotemporal control. Many PPI detection methods lack spatiotemporal control or an “on/off switch...outside the native cell context. The Split-BioID method Enter Split-BioID, a spatiotemporally controllable...complementation assay (PCA). In the Split-BioID method, each BirA* fragment is then fused to one of two...Split-BioID Split-BioID’s key advantage over other PPI methods, including BioID, is its spatiotemporal controlled...with the bait in a native cell contact. Other PPI methods may require protein interactions to remain stable...

...the CUT&RUN method, it seems likely that this technique will quickly become a standard method alongside ...high-throughput sequencing, ChIP-Seq, is the go-to method for mapping where a protein binds genome-wide, ...does have limitations. The enzyme-based CUT&RUN method overcomes many of these limitations and makes it...non-specific or indirectly interacting. The CUT&RUN method Enter CUT&RUN, an enzyme-based protocol for identifying...high-throughput sequencing. Improvements to the CUT&RUN method and using it in your own lab Very recently, the...

...as compared to spectrophotometric methods that measure all nucleic acids). These methods are more sensitive... is something that absorbance based methods can’t tell you and you don’t need a spectrophotometer or a...sequencing. Unlike absorbance-based methods, fluorescence-based methods require a standard curve, a set of...piece of DNA. This method is best used for DNA fragments (like a PCR product). This method also gives an indicator...bands or streaking. Unlike absorbance based methods, this method doesn’t tell you about contaminating proteins...standard plasmid prep. Diphenylamine method Another absorbance based method for quantifying DNA uses diphenylamine... advantages and disadvantages of each method and when one method might be more suited than another. ...

...schematic of CRISPR/Cas based COVID-19 testing methods.   Rapid detection of 2019 novel coronavirus...test that has been developed so far.  A similar method has been optimized to detect the SARS-CoV-2 virus... more details on the differences between these methods, check out this table.  All-in-one dual CRISPR/...Approach :This preprint uses a previously developed method to detect SARS-CoV-2 in the samples. First, RNA...regions, PAM can be engineered into the primers. This method does not depend on collateral cleavage by the enzyme...target regions thus making it an affinity-based method. Due to the mismatch sensitivity of FnCas9, this...portable coronavirus SARS-CoV-2 sequence detection method based on CRISPR-Cas12. Cold Spring Harbor Laboratory...

...consideration is: The number of inserts ...as some methods are more amenable to combining multiple DNA fragments... a high-level comparison of popular cloning methodologies, with a special emphasis on these five categories...Please note that many of the following cloning methodologies have been extensively described in other articles..., Golden Gate cloning might be for you. This methodology relies on versatile Type IIS restriction enzymes...option to consider when time is of the essence. This method requires a vector backbone containing a topoisomerase...choice, because the T4 polymerase needed for this method is relatively inexpensive and can be obtained for...approach, you might want to give Gateway a try. This method, developed by Invitrogen and based on lambda phage...

...cause demethylation?  Dividing cells depend on DNA methyltransferase 1 (DNMT1) to copy methylation marks...slightly smaller population, something else was also suddenly cool: DNA methylation. In 1983, Andrew Feinberg... proteins are not demethylases; they are dioxygenases that oxidize 5-methylcytosine to create rare DNA...complete DNA demethylation can be achieved by dCas9 alone, without TET (or any demethylase) fusion. This...site. So, when DNMT1 methylates the genome during DNA replication, it doesn’t methylate sites blocked by ...the original methylated strand is diluted to undetectable amounts: ergo, DNA demethylation! With optimization...optimization, this method can produce nearly 100% demethylation without the confounds inherent to dCas9-TET...

...Nielsen lab’s CRMAGE system is a fast, multiplexable method that combines CRISPR and recombineering-based MAGE... template or non-template strand, among other parameters. .  Stanley Qi lab's CRISPRi plasmids Stanley...based CRISPRa In the search for a CRISPR activation method in E. coli, Jesse Zalatan’s lab used an approach...as a collection of organisms, their genes, and metabolic processes, occupying the gut. Many human diseases...bacterial strain introduced to the population as methods of introduction. Engineering the bee gut microbiome...