...antibody you need will be conjugated, or that it will be conjugated to the conjugate of your choice. Knowing...go over some of the common conjugates you may encounter and how conjugates and antibodies come together...more, common conjugate class than fluorophores are enzymes. The most common enzyme conjugates are Horseradish... antibody-oligonucleotide conjugates used in assays like proximity ligation, immuno-PCR, and single-cell...purposes.  Coupling up - antibody conjugation chemistry  Whatever the conjugate, the molecules must somehow ...available conjugated antibody will have been verified to perform the same as its unconjugated version, ...hard to see. To solve this issue, researchers conjugate various labels to antibodies that produce detectable...

... the Gateway cloning method and its advantages. An introduction to Gateway technology The Gateway cloning...one Gateway vector to produce the desired expression clone. This is possible thanks to the Gateway vectors...Systems (MuLE) Kit, the MultiSite Gateway cloning kit, and MultiSite Gateway plasmids. Constant reading frame...positive (antibiotic) and negative (CcdB) selection markers used for Gateway Cloning can increase cloning...links below to find Gateway plasmids for your organisms of interest: Find Gateway cloning plasmids here... needs. Since its invention in the late 1990s, Gateway cloning technology has become very popular as a...appropriate entry and destination vectors, one can use Gateway to clone a gene of interest into a variety of expression...

...Joanne's Reddit AMA   Benefits of delegating The bottom line – delegation allows you to get more done. There...do. You can’t delegate the giving of feedback or performance review. You can’t delegate tasks assigned...what is to be delegated to them. Not all tasks are fun, but getting buy-in when delegating can help ensure...Aren't You Delegating" in the Harvard Business Review. Another excellent blog on delegation tips and practices...successful in your role if you become a master at delegating tasks and responsibilities. The manager is not...host of other advantages to practicing effective delegation skills: The team will be stronger and less ...doesn’t seem to have enough to do…you are not delegating enough. For example, if we need to develop a ...

...planning to conjugate your antibody in the lab, you’ll therefore want to research your conjugation reaction... to amine groups present in some conjugates, blocking the conjugation reaction. Many commercial antibodies...antimicrobial.  Conjugates And HRP brings us handily to the final topic of this post, the conjugates, or signaling...considerations, and give just the lightest of nods towards conjugates.  Storage   Once you get your exciting new antibody... as you move forward:  Was a light-sensitive conjugate on the antibody left exposed to light?  Has this... from the buffer and then re-added after the conjugation reaction is done, through a variety of methods...methods or commercial kits.  However, antibodies conjugated to horseradish protein (HRP), primarily used in...

...amplification (Option 2). Advantages of Golden Gate cloning Golden Gate cloning is one of the easiest cloning ... result, the ligation process is close to 100% efficient. Another strength of Golden Gate cloning is its...method, but can be assembled with Golden Gate. The popular Gateway cloning system produces constructs with...amino acids, but Golden Gate assembly can be designed to be scarless. Golden Gate assembly is also less ...Golden Gate and synthetic biology Synthetic biologists have leveraged the power of Golden Gate cloning...multiplexing easy.  Disadvantages of Golden Gate cloning Golden Gate cloning is not 100% sequence-independent... result in unintended ligation products.  Whether you are using the Golden Gate method to create CRISPR...

...recognizing a broad range of PAMs like NG, GAA, and GAT, but also displaying increased editing specificity...showed the broadest PAM range, recognizing NG, NNG, GAT, and CAA PAMs, while xCas9 3.6 displayed the second...at an NGG PAM. When they tested a variety of NG, GAT, GAA, and CAA PAMs, they saw that xCas9 3.7 cleaves...efficiency), NGC (2.1-fold), NGA (1.6-fold) and GAA and GAT (5.2-fold). The low increase in NGA editing efficiency...activating transcription. Sites containing NG, GAA, or GAT PAMs averaged 56-91% transcriptional activation compared...28%). Cytidine base editing at NGT, NGC, GAA, and GAT PAMs is improved, with a smaller increase in NGA ...containing ABE7.10, as well as improvements at NG and GAT PAMs. xCas9 3.7 Activity in Mammalian Cells Construct...

...first three gates will likely be: the FSC/SSC gate to identify cells, the single cell gate to exclude ...cell gate. However, make sure to compare H and A (or W and A).   Live/Dead Gating Having gated my single...quadrant gate, I am free to use multiple individual gates. Note that in this case, square gates were used...Live/Dead gate to focus your analysis on what’s biologically active. After that, the next gates fully depend...restrictive gate results in fewer cells being considered for the analysis. (C) A more liberal gate allows ...Depending on the following gates, cells that are not of interest can still be gated out later. Based on...make stricter gates while some like to make looser ones (Figure 3). A more “liberal” gate in the beginning...

...In a lab, you may have heard the phrase gram negative or positive being used to describe a species of... as gram positive, and the decolorized as gram negative. A German scientist, Carl Weigert, later expanded...1983. The differences between gram positive and negative bacteria is due to differences in cell wall composition...bacteria include staphylococci and streptococci. Gram negative bacteria, or diderms, have a thinner peptidoglycan...membrane and an outer lipid membrane. Common gram negative bacteria include salmonella and E. coli. Because...membrane, while the stain is washed from the gram negative bacteria’s thinner membrane. Procedure The stain...bacteria will be stained blue or purple, and gram negative bacteria will be stained red or pink, as shown...

...Lohmann said. The key component of Golden Gate and GreenGate are type IIS restriction endonucleases. They...breakthrough in cloning technology with the Golden Gate method. For the first time, it was possible to quickly...inexpensive enzymes. Now, a new toolkit - aptly named GreenGate – offers all of those same advantages to researchers... your target organism. We have designed a Golden Gate system based on the daily needs of an advanced molecular... molecular plant science lab.” Introducing GreenGate As Lohmann and his colleagues describe in the PLoS...ONE paper that introduced the kit in December, GreenGate includes the six most frequently used elements... selection of transgenic plants. The complete GreenGate toolkit, now available at Addgene, also gives ...

... on size is ultracentrifugation. Sedimentation equilibrium analytical ultracentrifugation yields an affinity... series, we will discuss how to select a non-aggregating fluorescent protein. In the jellyfish Aequorea... affinity and a tendency to form higher order aggregates. This property can be tolerated in some applications...measure your fluorescent protein’s propensity to aggregate. The basics and pitfalls of these experiments ...experiments are discussed here. In vitro tests for aggregation The tendency of purified fluorescent proteins to...homodimerization in aqueous solution. In cyto tests for aggregation To what extent does homodimerization assayed ...correctly to gap junctions (Shaner et al, 2008). Obligate dimers (such as dTomato) that promote interactions...

...transduction, conjugation, and transfection. Transformation, transduction, and conjugation occur in nature...our Viral Vectors 101 eBook. Bacterial conjugation Conjugation was the first extensively studied method...type E. coli (Griffiths et al., 2000). During conjugation, genetic material is transferred from a donor...such as antibiotic resistance. Bacterial conjugation as explained by the "I made this" meme! Comic...molecules so that it can more easily cross the negatively charged cell membrane. Physical methods such ... AJF, Miller JH, Suzuki DT, et al. Bacterial Conjugation. In An Introduction to Genetic Analysis. 7th ...

...your research! The starting point: Ligation-independent cloning Ligation-independent cloning (LIC) was first...methods had personalities, SLIC (sequence- and ligation-independent cloning) would be a true rebel. Not...site-specific recombination, it also doesn’t require a ligation step. Based on the robust system of homologous...through transformation, so the cloned vector must be ligated. But in LIC, longer single-stranded overhangs can... to specially designed plasmids. Sequence- and ligation-independent cloning: A SLIC makeover In 2007, ...Stephen Elledge. His new method, named sequence- and ligation-independent cloning (SLIC), eliminated many of...the use of another cloning method, such as Golden Gate assembly, may also be beneficial. SLIC is most often...

...Golden (Gate) pot (Pryor et al., 2022).     Figure 1: Workflow for high complexity Golden Gate Assemblies...upper limit for Golden Gate Assembly size. Ultimately, data-driven Golden Gate Assembly greatly increases...If you’ve ever used Golden Gate Assembly for cloning, you might be familiar with the rules of thumb for...published a paper profiling four base overhang ligation (Potapov et al., 2018), which showed that the ... to help researchers design high-fidelity Golden Gate Assemblies and overhang sets: NEBridge Ligase Fidelity... NEB successfully performed a 35-fragment Golden Gate reaction, using four-base overhangs, in a single...design rules) for successful high-complexity Golden Gate Assembly reactions. NEBridge Ligase Fidelity Viewer...

...to these efforts is the open sharing of negative data.  Negative AAV data is highly desirable  In a survey... respondents indicated they wanted to see more negative data (Figure 2.) At the same time, when scientists...don’t share, many express concern about sharing negative data they cannot explain, perhaps out of fear ...may not be the case. We can all acknowledge that negative data is something every researcher experiences...actively work to remove the stigma around sharing negative data and instead focus on getting this information...data. So take advantage of it and get your data - negative AND positive - out into the public eye where it...that contribute to recombinant AAV2 particle aggregation and methods to prevent its occurrence during ...

... selection, negative selection, and/or screening. We’re focusing on positive and negative selection in...effect. Negative selection is sometimes called counterselection.  Toxin-antitoxin systems One negative selection...but others will uptake plasmid backbones that re-ligated back on themselves. Therefore, your cloning strategy...containing 23 plasmids to complement auxotrophy. Negative selection: Cells that have lost a specific gene...gene survive Unlike positive selection, negative selection means you’re selecting for the loss of a gene...selection strategy uses the toxin CcdB. Gateway cloning takes advantage of this property by including ccdB...converts sucrose into a toxic metabolite in gram-negative bacteria. Plating on sucrose medium will select...

...we introduce you to the Journal of Emerging Investigators, an open-access journal that enables high school... the interview with the Journal of Emerging Investigators Podcast breakdown Intro Chapter 1 - Introduction...Olivia Ho-Shing, and the Journal of Emerging Investigators. Chapter 2 - Getting high school students and...

...Biolabs. Golden gate assembly limitations Embraced by the synthetic biology community, Golden Gate Assembly ...omitted. Screening for successful Golden Gate Assembly All Golden Gate assemblies feature an inverse proportionality...mismatch-containing pairings. During Golden Gate assembly, ligation of mismatched pairs of overhangs can lead...overhangs for any Golden Gate assembly design. Number of fragments assembled Golden Gate Assembly protocol...al. "Optimization of Golden Gate assembly through application of ligation sequence-dependent fidelity ...with both robust efficiency and accuracy. Golden Gate assembly involves the simultaneous activities of... the overhangs of the assembled inserts. After ligation of the insert into the vector, the BsaI site is...

...variant  3' NGAN or NGNG  xCas9 3' NG, GAA, or GAT SpCas9-NG 3' NG  Staphylococcus aureus (SA); ... 3' NNNNRYAC  Neisseria meningitidis (NM) 3' NNNNGATT  Streptococcus thermophilus (ST) 3' NNAGAAW...

...SpCas9. xCas9 has a PAM requirement of NG, GAA, or GAT, making it a more flexible cutter with the added ...