...depths (1 mm), important for certain applications such as in vivo tissue imaging.   Figure 1: Ace-mScarlet.... They can be targeted to the plasma membrane to measure membrane potential and can also be directed to... the membrane), the voltage sensing component (i.e., rhodopsin naturally resides in the membrane), or ... of voltage gated ion channels all depend on transmitting and sensing electrical potential. Thankfully... want to monitor the voltage change across the membrane of cardiac cells. Similarly, neuronal signaling...propagated through multiple cells to do things like translate perceived signals to physical actions. Understanding...rhodopsin proteins were initially evolved for ion transport, not fluorescence, they are not nearly as bright...

...interrogated by the optical system (Figure 1).   Figure 1: Cells expressing a particular surface ...populations of cells in a complex tissue or sample (1). When combined with cell sorters, this technology...permeabilize the cell membrane to allow antibodies to pass through the cell membrane. Formaldehyde fixation...dye emits green, yellow and orange photons while PE emits yellow and orange photons. If FITC and PE were...sources that allow users to assess a cell’s size, granularity, and target protein composition. It is used for...target protein expression levels, assessing post-translational modifications, determining cell health, analyzing...a pressurized buffer called sheath fluid and transported through tubes or capillaries to a laser. As the...

...detects samples at clinically relevant concentrations (1-3 fM; 1x106-fold lower than before), and our combined... health, energy, and sustainability.   References 1. Pardee, Keith, et al. "Rapid, Low-Cost Detection ...fast-tracked development of diagnostics. Mostly transmitted by Aedes mosquitoes (aegypti and albopictus) ...and electronic companion technologies that has transformed the assay’s capabilities. Specifically this includes...

...Updated Jun 1, 2021 by Meghan Rego. Reproducible data are key to science, so scientists are used to repeating...the DNA packaged inside the viral particles (Figure 1). Briefly, packaged DNA is extracted from purified...serotype. Additionally, we check viral transgene expression in transduced cells whenever possible. This post... for transgene expression. Once packaged in AAV, this pre-recombined DNA could lead to transgene expression... to these expected hits, we always get hits to “random” genes. Do these hits always mean the sample is...wanted to see how low we could go. We prepared a range of spiked samples from 0.1-20% and discovered that...find the optimal dose that allows for sufficient transgene expression but limits the likelihood of recombinase-independent...

...fluorophores are constantly being engineered. Table 1 lists a few common fluorescent proteins and their ...definitions found in Table 2 may not apply. Table 1: The specific mutations comprising common fluorophores...physiological processes. Re-engineering GFP to increase its range of color and application Almost as soon as its...backbones for constructing fluorescent fusions. Transcription Reporter: Placing GFP under the control of a...promoter in a given cell type. This type of transcription reporting was among the earliest uses of GFP...GFP. Fluorescence resonance energy transfer (FRET): This is used to study the interactions between two ...ion (such as Ca2+) concentrations and pH, using a range of strategies such as FRET, calmodulin, and others...

...In Biochimica et Biophysica Acta - Biomembranes (Vol. 1838, Issue 1 PARTA, pp. 15–33). https://doi.org...principles of fluorescent dyes/ligands Figure 1: Example structures of fluorophores, fluorescent dyes... dopamine D2/D3 receptors. Scientific Reports, 10(1). https://doi.org/10.1038/s41598-020-78827-9 Breen... of fluorescence: For applications that involve ranges of pH, and significant lengths of time, the stability...receptor conformation and signaling activity​​ (Sridharan et al., 2014). It's also crucial to evaluate how...the background. Dyes that can pass through cell membranes can prove useful for live-cell imaging. Experimental...considerations regarding the choice of fluorescent dyes (Sridharan et al., 2014). Manufacturers will typically recommend...

...for example, split cells on a Monday diluting them 1:10, you should be able to split on the following Thursday...Immunology. You can find Sana on LinkedIn.   References 1. Yin, R., et al. "Gene expression profiling analysis... are sensitive to pH. Anything between orange and light orange usually indicates that the cells have grown...culture media contain the pH indicator phenol red (pH range 6.0 (yellow) - 8.0 (red)). Blood pH normally lies...too young and should not be used for important transfection experiments right after they’ve been thawed.... spent organizing, an hour is earned (Benjamin Franklin)! It’s good practice to label your plates and/...

...CRISPR tools.  Distributing and depositing In Figure 1, you can see cumulative numbers of CRISPR plasmid ...new CRISPR depositors each year.    Figure 1: Addgene CRISPR distribution and deposits. (A) Cumulative...generate a classic double-stranded break, while modulators regulate transcription and chimeric efforts make...Countries shown are the top 10 requesting countries. FR=France; KR=South Korea; CH=Switzerland; AU=Australia.  ...Pyhtila et al., 2023). Our thanks to Rodolphe Barrangou for his authorial contributions to, and support...References Pyhtila, B., Kasowitz, S., Leeson, R., & Barrangou, R. (2023). The Expanding Dissemination and Distribution...

...listen to the interview with Tom McFadden!   1. Rosalind Franklin vs. Watson & Crick - Science History Rap...telling following the unfortunate story of Rosalind Franklin and her incredibly important role in solving the...

...cleavage (Harrington et al., 2017). Figure 1: Anti-CRISPR proteins (Acr's) can block CRISPR activity... ways to anti-CRISPR proteins in your experiments 1. Reduce off-target effects Prolonged Cas activity ... Knott GJ, Edraki A, Garcia B, Amrani N, Chen JS, Cofsky JC, Kranzusch PJ, Sontheimer EJ, Davidson AR,...695 . https://doi.org/10.3390/v10120695 Pawluk A, Amrani N, Zhang Y, Garcia B, Hidalgo-Reyes Y, Lee J, Edraki...

...editors (ABE) mediate A•T-to-G•C base changes (Figure 1), but it can be challenging to make these base changes...dCas to the genomic DNA of interest  Figure 1. When ABEs are in action they bind DNA so that the...the TadA domain can edit the short single-stranded DNA stretch that is accessible. Image from Gaudelli ... protospacer, the editing capacity of the ABE8s ranged from 1.5x higher for positions A5-A7 to 3.2x higher...References Gaudelli NM, Komor AC, Rees HA, Packer MS, Badran AH, Bryson DI, Liu DR (2017) Programmable base ...

...10.1073/pnas.0909910107 Varani G, McClain WH (2000) The G·U wobble base pair. EMBO Rep 1:18–23 . https://doi.org... cerevisiae (Tuller et. al., 2010). Figure 1: An overview of peptide synthesis. A ribosome interacts...is codon usage important? Translation and protein synthesis relies on transfer RNAs (tRNAs). tRNAs bind...rate of translation and thus protein expression. In fact studies have shown that translational efficiency...on an organisms codon usage bias to increase translational efficiency and thus protein expression without...mammalian cells. This was the first codon optimized membrane protein expressed in mammalian cells. In this ...conformation, folding and stability, change post-translational modification sites and even affect protein function...

...advancing the field. References: 1. Internal initiation of translation of eukaryotic mRNA directed by a... of a 2A peptide derived from porcine teschovirus-1 in human cell lines, zebrafish and mice. Kim et al...discovered in positive single-stranded RNA viruses to allow for the efficient translation of multiple genes from...Elements Translation in eukaryotes usually begins at the 5’ cap so that only a single translation event ...scientists use a multitude of techniques including co-transfection of two or more plasmids, the use of multiple...Unlike promoters which will create unique mRNA transcripts for each gene that is expressed, multicistronic...your gene as they are both derived from the same transcript. Of course multicistronic vectors do not have...

... depending on the specific edit.   Figure 1: Choosing the best PE6 variant for your application...modular envelope design. Nature Communications, 14(1), 5141. https://doi.org/10.1038/s41467-023-40788-8...also incorporated LOV-Turbo into TransitID experiments.  Find TransitID plasmids here! These two developments...genetic cargo Optogenetic proximity labeling and TransitID Tracking cellular history with protein ticker ...in vitro and in vivo. Incorporating reverse transcriptase (RT) variants, PE6a and PE6b are 500–800 bp ...be tethered on the viral surface, expanding the range of targetable cell types. DIRECTED is compatible...37612276.   Optogenetic proximity labeling and TransitID By Mike Lacy Proximity labeling can identify protein...

...systems reviewed here. The only required components are 1) a SMASh-tagged protein and 2) asunaprevir (small ... tag size, which can also be handy!   Fig 1. dTag molecules induce dimerization of an FKBP12F36V...of an exogenous small molecule). Degron tags can range in size from several kDas up to ~35 kDa and are ... because the system modulates protein post-translationally, unlike many other methods. Secondly, these...

...sequence derived either from p63(1-29) and Lck. Image from Chertkova et al., 2020. In their BioRxiv post...Reports. 2020. https://doi.org/10.1038/s41598-020-57500-1 The CRISPR corner By Jennifer Tsang Here are a few...protease allows a transcription factor to enter the nucleus and modulate transcription of the mCitrine ...HeLa cells were transfected with plasmids encoding FPs tagged with the plasma membrane targetin sequence...non-specific labeling. For labeling the plasma membrane, they tested different target sequences for improved...expression can vary significantly depending on transgene design and delivery. To overcome these variations...into genomic DNA with the help of the piggyBa transposase. Image from Hazelbaker et al., 2020. The lab...

...be selected for these small ligands. References 1. Ma, Hanhui, et al. “Multicolor CRISPR labeling of...Biological Applications.” Sensors (Basel). 2012; 12(1): 612–631. PubMed PMID: 22368488. PubMed Central PMCID...fluor. Transcribing and translating a protein fluorophore also takes time - bypassing translation altogether... could be screened for their affinity to a wide range of non-nucleotide targets. These RNA molecules, ...The key to aptamers’ flexibility is their broad range of targets, including proteins, peptides, amino ...ions, and cells (even pathogenic bacteria). That range is possible because aptamers can either fold to ...biosensors. Aptabeacons Fluorescence resonance energy transfer (FRET) technology has been studied and used in...

...Time for the #BioSci3319 #StreakPlateChallenge part 1. Lab B needs a redo :-( @SandTBioSci #CompetitiveStreaking...and spread over the second quadrant. The process continues until all quadrants are used, hopefully revealing...students transform them into different strains of E. coli,” says Westenberg. During the transformation experiment...plate, first the bacteria are inoculated over a quadrant of an agar plate. Then, a stick is dragged through...revealing single, isolated colonies in the last quadrant after incubation. The challenge is usually successful...genetic backgrounds of the host strains affect transformation efficiency.  Aside from the Streak Plate Challenge...

...existing protein structures, called Top7. Figure 1: Top7 receives the Nobel Prize. Created with BioRender.com...References Kuhlman, B., Dantas, G., Ireton, G. C., Varani, G., Stoddard, B. L., & Baker, D. (2003). Design...

... to the development of resistance include (Figure 1): Over-prescription of antibiotics (for example, ... communication, and public health.     References 1. O'Neill, J. "Tackling drug-resistant infections globally...methods of ‘horizontal gene transfer’. Resistance can also arise from random mutations in the genetic sequence...didn’t develop, either through random mutation or horizontal gene transfer, means to defend themselves from...bacterium sharing genes with another, an accidental transmission of genes from one bacterium to another through...

...true reproducibility. The following are critical: (1) connectivity to other data sources used in research...engagement with the Wisconsin research community were to: (1) prevent vendor lock in by making it easy to get data...that emerged from intensive engagement with a wide range of people at the University of Wisconsin in 2011...reproducibility an ELN should have integrations with a broad range of relevant generic and science-specific tools. ... however, in some cases it would be possible to grant those wanting to understand and/or attempt to reproduce...Massachusetts, and feels privileged to be part of the vibrant life sciences research and commercialization community...

... new preps might enhance your research!   Table 1: New ready-to-use viral vectors in the Addgene repository...diverse needs of our community and support a wide range of experimental designs. Here are nine great additions...Request service, which offers over 3,000 eligible transfer plasmids and five different serotypes. Not sure...

...more applications.   Fig. 1 Making a Protein Tag (Abridged) (1) Locate a promising monoclonal antibody...an amide group added to its C-terminus post-translationally, which turned out to be critical for antibody...

...does not greatly alter the size of the tag.   Table 1: Common protein tags Tag Epitope Mass (kDa) Function... conditions FLAG DYKDDDD or DYKDDDDK or DYKDDDK 1 Affinity and Purification Good for antibody-based ...usually smallish peptides incorporated into a translated protein. As depicted in the accompanying cartoon...peptide-based proteosome inhibitors, glutathione S-transferase (GST), which can be fused to recombinant proteins...

...dark affinity range (130nM to 4.7µM for iLID nano and 800nM to 47µM for iLID micro; Figure 1). With this... August 20th; 14(1). TRICK: A method for visualizing the first round of translation in living cells ...light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2). In the dark, this peptide is caged by the...Figure 2). They are all available now at Addgene. 1 Lungu et al., Chem Biol. 2012 Apr 20;19(4):507-17....Guntas et al., Proc Natl Acad Sci USA 2015 Jan 6; 112(1):112-7. doi: 10.1073/pnas. CRISPR-Cas9 optogenetic...for a variety of experimental purposes including (1) single or multiple, constitutive or inducible, gene...the years. The authors optimized these systems by: 1) decreasing the number of plasmids required for each...

...fragments of these transcripts. I remember when small RNA was just something we ran off the bottom of ...viruses - 2 cas systems regulate at the DNA level and 1 at the RNA level. Udi Qimron from Tel Aviv University...’t give tRNAs much thought. High throughput transcriptome sequencing methods including work done by the...hypothesized that life started with RNA and a simple cell membrane. His recent work shows that they are making fairly...knew…I had barely heard of these). These single stranded, covalently closed RNAs seem to be conserved in...

...single neurons in the living brain   Figure 1: Plasmid mix to label neuronal morphology (eYFP) and...genOway press release for more information. References 1. Atasoy, D., Y. Aponte, H. H. Su, and S. M. Sternson...concentration of FLEx vectors guarantees that most cells are transfected with all labeling plasmids and...PMCID: PMC2593125. 2. Buchholz, F, P O Angrand, and A. Francis Stewart. 1998. “Improved Properties of ... sites can lead to excision, inversion, and translocation of DNA in a similar fashion as between loxP ... has a temperature optimum of 37 °C (Buchholz, Angrand, and Stewart 1998). FLPe’s performance was further...in FLPO, which shows similar efficiency to Cre (Kranz et al. 2010). These differences in recombinase efficiency...

...Addgene's founders Podcast breakdown Intro Chapter 1 - Who are Addgene's Founders? Chapter 2 - The process...Chapter 3 - Addgene as a leader in standardized tech transfer and the adoption of the UBMTA Chapter 4 - Developing...ensuring that success Chapter 6 - Developing Addgene’s Brand and culture Chapter 7 - Building Addgene as a global...

...Figure 1). Clustered events are called a population.                             Figure 1: While...conceptualize where the populations are, try drawing a quadrant over the plot that divides it into four, using...different populations by imagining (or drawing) a quadrant over the dot plot. Created with biorender.com....Biorender! While a helpful visualization tool, a quadrant will often be too clean for real-world data. Most...and debris. The side scatter roughly measures granularity, a proxy for nucleic complexity of the cell.  ...these populations, I suggest using the imaginary quadrant to help (Figure 5). Even though this real-world...real-world data is far messier than biorender plots, quadrants can still help me as a reader if I'm struggling...

...deficient in DNA cutting activity to a transcriptional activation domain (Fig 1). Two ways to use CRISPRa for... (2016) Targeted HIV-1 Latency Reversal Using CRISPR/Cas9-Derived Transcriptional Activator Systems. PLoS...promoter of another gene, thereby driving aberrant transcription. That being said, we generally pick guides...been significant work on constructing Cas9 transcriptional activators. These constructs allow for the ...picking whichever guides are closest to the transcription start site (TSS). We recommend targeting the...Church GM (2015) Highly efficient Cas9-mediated transcriptional programming. Nat Methods 12:326–328 . https...Nishimasu H, Nureki O, Zhang F (2014) Genome-scale transcriptional activation by an engineered CRISPR-Cas9 complex...

...Spaghetti Monster probes at Addgene Uses of smFPs 1. Multi-channel connectomic tracers Fluorescent proteins... expanded use of smFPs in the future. References 1. Viswanathan, Sarada, et al. "High-performance probes...ideal probe should combine the solubility, cell tolerance, and optional fluorescence of FPs with facile ...

...know that most cephalopods die after they lay their 1 clutch of eggs? Propagation is thus quite difficult...organism blog posts: Five Popular Model Organisms, Part 1 Five Popular Model Organisms, Part 2 ...species. Discovering ways to culture, propagate, transform, store, and share all of these new organisms continues...Phenotype thus making progress on the NSF “G to P grand challenge.” Creating a productive, new scientific...evolution because electric organs arose in different branches of the evolutionary tree at least 6-7 times and...mushrooms) but there are over 1,000 species. No transformation techniques are available yet. Polistes (Amy... a system using novel vesicles to efficiently transform these cells which don’t like foreign DNA at all...

...folding, and deal with sub-optimal codon usage (Table 1) A few mutations are common to all or most expression...curated list of Bacterial Expression Systems Table 1: E. coli expression strains  Note: All strains are...specialized strains are available to confer greater transcriptional control, assist with proper protein folding...harboring this mutation are deficient in outer membrane protease VII, which reduces proteolysis of the...Lacks functional RNaseE which results in longer transcript half-life F- ompT lon hsdSB (rB- mB-) gal dcm...Chloramphenicol (pRARE) Good for “universal” translation; contains 7 additional tRNAs for rare codons ...pLemo (CamR) Expression of toxic, insoluble, or membrane proteins  T7 Express   IPTG-inducible expression...

...sequence on either side of the desired mutation (usually 1-3 mismatched bases) is sufficient for your primers...researcher at Cornell University.       References 1. Breslauer, Kenneth J., et al. "Predicting DNA duplex...homology-directed repair (HDR) of a CRISPR/Cas9 induced double-stranded break. This site-directed genome editing requires...methylation-dependent endonuclease (i.e. DpnI), and bacteria are transformed with the nuclease-resistant nicked plasmid (the...products of the PCR reaction are pieces of double stranded, circular DNA with staggered nicks, recent data...final PCR products are pieces of linear, double stranded DNA with homologous ends. These homologous ends...the adenosine of the GATC recognition site. Transformation: After the PCR reaction, no ligation is required...

...$47,476 and the date for implementation was set for Dec 1 2016. In order to be exempted from the need to pay... these institutions (populations potentially from 1 to 5761, according to the NSF data) and therefore ...about? In March 2014, the White House issued a memorandum to the Secretary of Labor to update the FLSA,...postdocs in the final ruling was, for a lot of us, frankly quite surprising. Consortia of higher education...issued, a joint blog post by Director of the NIH Francis Collins and Secretary of Labor Thomas Perez, Fair...exactly how postdoctoral salaries are changing. Transparent salary information for postdoctoral positions... of postdocs at different points on this salary range. We have been able to gather data on changes to ...

...possible without double-strand breaks (DSBs) or donor DNA templates.  Figure 1: An overview of prime...demonstrated ~20-50% efficiency in HEK293T cells with 1-10% indels. Since then, further innovations have continued...Engineering. https://doi.org/10.1038/s41551-024-01227-1 Yan, J., Oyler-Castrillo, P., Ravisankar, P., Ward... non-edited strand, they can cause the cell to remake that strand using the edited strand as the template...the nicked target strand. The initial result is a heteroduplex, with overlapping strands of edited and unedited...Improving reverse transcriptase efficiency Building upon prior reverse transcriptase research, the Liu...the edit into one strand, there’s a mismatch between the original sequence on one strand and the edited ...

...different ways: Transformation grade DNA Cloning grade DNA Transfection grade DNA Figure 1: Transformation... differences between transformation, transduction, conjugation, and transfection Resources on Addgene.org...Transformation introduces transformation grade plasmids into a bacterial cell. Transformation grade DNA  Transformation...bacterial transformation of chemically and electrocompetent cells is minimal (picogram range). It can ...plasmids into mammalian cells? Use transfection grade DNA. Transfection is the process of inserting an ...without commercially available kits and DNA for transformation doesn’t need to be as pure as for other uses...scientists to clone from a smaller amount of DNA (ng range). To allow efficient and flawless cloning it is ...

...forward to another great year of sharing!   References 1. Shalem, Ophir, et al. "Genome-scale CRISPR-Cas9 knockout... name suggests, lentiCRISPR v2 is a lentiviral transfer vector used to deliver both Cas9 and your gRNA...metalloproteinase-9 secretion, and identifying lysine methyltransferase substrates. We’re incredibly happy that lentiCRISPR...

...is here to help. Contact us at [email protected] or +1 617-225-9000, or check out our MTA reference pages...Uniform Biological Material Transfer Agreement (UBMTA) for its material transfers. The UBMTA was drafted by...organizations Assurance that recipient may not use the material for a commercial purpose Assurance that recipient... has time for paperwork? Here’s why a Material Transfer Agreement (MTA) matters: Science thrives on collaboration...universities and institutions require them for material transfers. When Addgene entered the plasmid sharing space...receiving institution. Therefore, many technology transfer officers are familiar with the UBMTA and are happy...  material transfer agreement...

...’ve come to the right place. Get started with PGC-1 plasmids from Addgene. References: Skeletal muscle...PGC-1a1, a transcriptional coactivator induced in skeletal muscle by exercise and endurance, protects ...have reported a mechanism based on studies of transgenic mice that helps to explain how it works. The ... skeletal muscle expression of kynurenine aminotransferases, to enhance the conversion of kynurenine into...

... successful after their training.   Figure 1: Professional Development Hub Workshop Attendees and...ample resources and a positive perspective on the transition to a post-academic career. This training was ... inadequate job of supporting science trainee transition into diverse, successful careers.  We’re not ...components of predoctoral and postdoctoral training. A grant from the National Institute of General Medical Sciences...

... specific post-transcriptional regulation of endogenous RNA. J Biol Eng. 2014 Mar 1;8(1):7. doi: 10.1186...ability to direct enzymes – from nucleases to transcription factors – to specific sequences of DNA has become...Biological Engineering in March is fused to a translational repressor. In principle, however, designer PUF...

...improved sensors for these and other targets. Figure 1: MTRIA biosensors are generated by inserting a circularly-permuted...fluorescent biosensors. Nat Commun. 2022 May 25;13(1):2919. doi: 10.1038/s41467-022-30685-x. Feel the ...new class of sensors for neuropeptides and neurotransmitters. Motivated to develop a biosensor for oxytocin...devised a “Modular fluorescence unit fused with TRansmembrane region-to-IntrAcellular loop linkers” (MTRIA...sensors based on GPCRs recognizing various neurotransmitters, neuropeptides and other ligands - almost ...kinetics and localization for imaging synaptic transmission. bioRxiv 2022.02.13.480251. doi: 10.1101/2022.02.13.480251...2022.02.13.480251 JEDI-2P voltage sensor The François St-Pierre lab and colleagues have developed a new...

...infections, cancer, and other diseases. References 1. Harrington, Lucas B., et al. "Programmed DNA destruction...that Cas14 could indiscriminately cleave ssDNA in trans like Cas13 and Cas12 do for RNA and dsDNA, respectively...can cleave fluorescent probes of dsDNA and RNA in trans. Fluorescent signal indicates the presence of this...

...education, government, and civilization in general (1). Science and politics? Science is an apolitical process...consequences of a march. Three of these risks include: 1) Lost trust Perhaps the greatest risk is that the ...working full-time, and have received a research grant in the last five years as polled in the American...climate change page on whitehouse.gov, freezing grants, and proposing to defund aspects of research at...following subjects: Climate change Vaccines GMOs Transgender issues Reproductive health Immigration Pollution...while many also seek to honor and credit women, immigrants, and minorities who have had great impacts on...encouraged by “Broader Impacts” requirements for certain grants) (28). As one young scientist, Matthew Niederhuber...

...fluorescent in acidic organelles (Figure 1), showing that its acid tolerance is maintained in cells.   GFP is ...References 1. Zacharias, David A., et al. "Partitioning of lipid-modified monomeric GFPs into membrane microdomains...of different fluorescent protein are available (Cranfill et al., 2016 and FPbase.org). A number of variants...specific RNA stem loop-structure (Querido and Chartrand, 2008). Finally, a nuclease-deficient Cas9 can...clearly the case when proteins undergo post-translational modification with fatty acyl chains. For instance...28360214. PubMed Central PMCID: PMC5385933. 6. Cranfill, Paula J., et al. "Quantitative assessment of ...PMC2644460. 12. Querido, Emmanuelle, and Pascal Chartrand. "Using fluorescent proteins to study mRNA trafficking...

...by insertion and deletion frequency. Fig. 1: Overview of TIDE. The input is sequencing trace files...keep your product to knock-in size ratio below 10:1. Small knock-ins Is your knock-in edit small, with...optimizing this step, or consider enriching for transfected cells by flow sorting or drug selection, assuming...

...with the Gibson assembly master mix for 1 hour, and then transform into competent cells. The success rate...you should consider giving it a try! References: 1. Gibson DG, Young L, Chuang RY, Venter JC, Hutchison...generating long overhangs which allows the single stranded regions with homology to anneal a polymerase, ...hour or so, the sample is immediately ready to transform into competent cells. The master mix of enzymes...if the ends of the fragments have stable single stranded DNA secondary structure, such as a hairpin or ...would directly compete with the required single-stranded annealing and priming of neighboring assembly ...

...at a low angle (FSC) or large angle (SSC) (Figure 1). FSC values depend on the cell's size, while SSC ... or ask experienced colleagues.   Figure 1: The laser pulse illuminates the cell and is scattered...software in order to generate signals in a reasonable range. If colleagues in your group already have experience...experimentally-specific gates. Two-dimensional staining (quadrant gating) For our last example plot, I will show...cells (CD8+) from dendritic cells (CD11b+) using a quadrant gate. Within my “lymphocyte + single cell + live... two stains in relation to each other. Using a quadrant gate, I can divide my cell population into DN,...for each stain respectively). (B) Instead of a quadrant gate, I am free to use multiple individual gates...

...compare the experimental group.     Figure 1: A, Most biological experiments will benefit from ... Histochemistry & Cytochemistry, vol. 59, no. 1, SAGE Publications, Jan. 2011, pp. 6–12. Crossref,...across samples and equal protein transfer from the gel to the membrane. A loading control is critical for...identically, used to assess naturally occurring random biological variability. If one were studying the...purple) and nonspecifically through the Fc region (orange). The Preimmune serum control will not bind to .... Always probe the loading control on the same membrane that the target protein was measured on.    Flow...