...chemically competent cells (protocol)  Preparing electrocompetent cells (protocol)  Untergasser Lab Preparation...chemically competent cells (protocol) Preparation of electrocompetent cells (protocol) At Addgene, we use...using the protocols provided above, then you can find the subsequent transformation protocols below: OpenWetWare...Transforming chemically competent cells (protocol)  Electroporation (protocol)  Untergasser Lab Transformation...chemically competent cells (protocol)  Transformation of electrocompetent cells (protocol)  Cells prepared ...streamlining the transformation step. Read on to find protocols and tips that you can use in your own lab. How...competent cells will work just fine.  A number of protocols for making competent cells are available online...

...studies. Endotoxin contamination is minimized by using an endotoxin-free plasmid purification protocol. To ... bacterial endotoxin is ensured to be less than five endotoxin units per mL. The endotoxin levels are ...recombination rates in FLEX/DIO/fDIO constructs. Endotoxin Endotoxin contamination in vector preparations can ...distributed to customers. Details about our production protocols, titering methods, and quality control are described...elements and an internal control of known titer (protocol modified from Lock et al., 2014.) Previous experience...vectors by optimizing and standardizing production protocols and performing rigorous quality control (QC). ...are determined using a chromogenic endotoxin detection assay based on the amebocyte lysate method. Purity...

...wavelengths of emitted photons are higher than those of absorbed photons. When photons excite electrons of... about 380–700 nm. Photons of lower wavelengths are higher in energy, while photons of higher wavelengths...whereby a photon is emitted. This step is called emission. During excitation, some of the photon's energy...the energy of an emitted photon is lower than the energy of the absorbed photon. Correspondingly, wavelengths...about the relationship between a photon's wavelength and energy, how photons can be absorbed and emitted by...700 nm make up the infrared (IR) spectrum. When photons are absorbed by matter, they promote electrons ... one defined wavelength. Likewise, the emitted photons also appear within a range of wavelengths. For ...

...Sacchi, 2006).  View our protocol for RNA extraction! Making the simple protocol even more foolproof (still...blog Read blog posts related to plasmid protocols Find protocols for purifying DNA without a kit Resources...Addgene.org Check out our molecular biology protocols Find protocol videos on our website Read our Molecular...   RNA extraction methods evolved into a simple protocol still used today There are many alternative methods... to that method. A major hurdle to developing protocols to isolate RNA was that RNases are commonly found...this (Chirgwin et al., 1979). They developed a protocol meant for isolating RNA from rat spleens in which...the NIH in the mid 1980’s set out to develop a protocol that skipped the ultracentrifugation altogether...

...Source of Emitted Photons Chemical reaction High-energy photons Kinetics of Photon Generation Slower...Modification Not Required Required Photobleaching/Phototoxicity Not susceptible Susceptible Subcellular... living organisms resulting in the emission of photons. The most familiar bioluminescent organism for ...accurate and safe. In the case of a luciferase assay, photon emission is measured resulting from the catalysis...luciferin, ATP and oxygen as substrates. Production of photons by this bioluminescent reporter occurs slower than... result of the different mechanisms to produce photons, chemiluminescent reporters are generally less ...background levels and improved signal sensitivity since photons are simply measured – they are not required to ...

...’ve shortlisted several protocols, including their phosphoserine ncAA protocol, to optimize and share,... posts on the various protocols and helping to spread the word as new protocols come out! References ...for all three components, meaning that the final protocol may involve a sensitive balance of trade-offs....development-focused field, it’s not uncommon to find protocols that require reagents which aren’t broadly or ...available, so you may find yourself wanting to use a protocol that you’ll have to rework to include reagents...between structure and function…if you can get your protocol to work. Or, to quote Ryan Mehl, PhD, GCE has ...resources, knowledge, and most importantly, validated protocols (using readily available reagents!) for the use...

...brightness, photostability, and most importantly, a photoswitching ability. This photoswitching refers to...fluorescence quantum yield, the ratio of photons absorbed over the photons emitted. For example, DAPI is much...Ghods, R. (n.d.). Comparison of the Photobleaching and Photostability Traits of Alexa Fluor 568-and Fluorescein... to an excited state through the absorption of photons. After a brief elevation in this excited state,...electron returns to its ground state by emitting a photon with slightly less energy due to non-radiative ...long-term experiments (Salari et al., 2019). Photostability, the ability to retain fluorescent intensity...longer periods of exposure, you’ll want a more photostable fluorescent protein (Mahmoudian et al., n.d.)...

...Tichy et al., 2019), photoswitchable tethered ligands (PTL), and photoswitchable, orthogonal, remotely-tethered...pathways? Here’s where photoswitchable intracellular proteins come into play. Photoswitches can be used to activate... G-protein coupled receptors (OptoXRs), and photoswitches. Light-gated ion channels directly open and ...potential and a host of other cellular functions. Photoswitches are engineered molecules that induce conformational...concentration gradient, and of activation inhibitory (ArCH) proton pump passing ions against the concentration gradient... special, covalently linked molecules (i.e. photoswitches) attached near the binding pocket of an engineered...activates downstream secondary signaling pathways. Photoswitchable tethered ligands hold the ligand in close proximity...

... to test several protocols that have been described (protocol 1, protocol 2, protocol 3) to find the one...system.   See our protocol pages for full Lentivirus Production and Transduction Protocols   Once purified...Most of the protocols you will find will recommend using a polycation, like polybrene (protocol) or DEAE ... to be taken especially if you are working with proto-oncogenes or with human samples that have not been...transfer vector. Lentiviral particles expressing proto-oncogenes or oncogenes for instance should be manipulated...order to precipitate the viral particles. Some protocols suggest adding sucrose to the supernatant to create...cells). Again, my tip here is to test several protocols as the set-up depends on the type of cell you ...

... As Nano-lanterns are not subject to photobleaching/phototoxicity, they successfully imaged multiple subcellular...long-term imaging, especially where photobleaching and phototoxicity are a concern, these constructs come...Long-term light exposure can also lead to cellular phototoxicity, and experimental success can be affected by...by both autofluorescence and photobleaching. Researchers have long been interested in using luminescence...bioluminescence resonance energy transfer (BRET). Photons emitted by a Renilla luciferase variant are used...in the culture medium to allow RLuc8 to produce photons. The original nano-lantern emitted yellow-green...Resources on the Addgene Blog Learn about Photoactivatable Fluorescent Proteins Check Out Fluorescent...

...substrate, luciferin, which releases energy as a photon. The photon energy is transferred to the acceptor fluorophore...range of mitochondrially targeted photoactivatable and photoswitchable FPs that are activated by UV light...subpopulations within the same cell; by photoswitching PSmOrange and photoactivating PAGFP in separate subpopulations...e43072. doi: 10.1371/journal.pone.0043072. Photoswitchable tools for spatial and temporal control of cell...Three years ago, Brian Kuhlman’s lab designed photoswitchable dimers by fusing the SsrA peptide with the ... light-sensitive LOV2 domain of Avena Sativa phototropin 1 (AsLOV2). In the dark, this peptide is caged...high throughput binding assays, to engineer photoswitchable dimers which show over a 50 fold change in ...

..., most of which is relatively cheap). Several protocols have been written outlining the different steps...steps and materials required for each [1, 2]. This protocol [3] provides, in exquisite detail, the various...the opsins will receive sufficient quantities of photons to become activated. Light is able to penetrate...between patch cable and implant. A steady stream of photons from the laser to the patch cable to the ferrule...ferrule implant must be maintained during the entire protocol, otherwise optical stimulation will be erratic...Channelrhodopsin 2 (Chr2) has been expressed in the motor cortex of a mouse. Upon connection of the ferrule...insensitive. If you deliver a continuous stream of photons, Chr2 will open but will not be given an opportunity...

...milk protocol is adapted from the 1979 Patterson Protocol. When published, the Patterson Protocol was ...plasmid purification protocol and guanidine isothiocyanate for the gel extraction protocol) disrupts the DNA...Product: plasmid DNA This kit-free plasmid miniprep protocol from Addgene follows a similar workflow as a column-based...iodine (NaI) solution. NaI has two purposes in this protocol: 1) it solubilizes both DNA and agarose, and 2...took advantage of this property and developed a protocol that uses Whatman No. 1 paper or even paper towels...our Plasmids 101 blog series Find other plasmid protocols and tips Browse our plasmid cloning blog posts...posts Resources on Addgene.org Find protocols for molecular biology and cloning Read our Molecular Biology...

...labs already familiar with IHC/ICC protocols. A typical ExM protocol (Fig. 1) follows this structure: ...steadily growing in popularity with an abundance of protocols and examples of applications from across disciplines...several times its original size. Depending on the protocol, samples can be expanded to ~4-10x linearly! This... using more accessible imaging equipment, ExM protocols utilize relatively inexpensive reagents and are... Steps 2-4 are not found in standard IHC/ICC protocols and you might expect them to dramatically limit...the types of labels you could use. But many ExM protocols are compatible with conventional antibodies, chemical...labels. You may want to check out a few different protocols to look for strategies that may be better for ...

...absorption of 540 nm and two-photon excitation around 1,040 nm, has larger photocurrents, and is more selective...they found an indicator that is optimized for 2-photon imaging of voltages. This includes monitoring of...resultant JEDI-2P sensors overcome many challenges in 2-photon in-vivo imaging of voltage fluctuations. Image ...recording using a fast indicator evolved for two-photon microscopy. Cell, 18, 3408 - 3425.e29. (2022). ...have high selectivity for K+ over Na+ and rapid photocurrent kinetics. They demonstrate the use of HcKCR1...frequencies with (magenta) and without (black) photoactivation of HcKCR1 in mouse neurons. D) Membrane voltage...mouse neuron without (top) and with (bottom) photoactivation of HcKCR1. Images adapted from Govorunova et...

...paper or protocol. If you have questions while reading these materials (maybe a step in the protocol you don...ask for a copy of the protocol if you prefer to take notes on a printed protocol, or maybe you would like...resources to help you get started!) Are there protocols you can familiarize yourself with? If your bench...bench mentor hasn’t sent you papers or protocols yet, don’t be scared to ask for some! You can email or...or “It would be great if you could send me the protocol we’ll be using next week so I can familiarize ...bare minimum should be taking notes on general protocol steps and other tips and tricks your mentor shares...next few days or weeks), look over your notes and protocol(s). Is there enough information there for you ...

... you will be able to confirm that the testing protocols elicited the expected response and have a baseline... of mice that is subjected to the experimental protocol but does not receive either protein kinase inhibitor...included to assess variability in the experimental protocol. In the experiment above, you could repeat the...knockout samples are treated with the identical protocols that the experimental samples undergo. If the ...repeated for each experimental run as long as the protocol remains the same.    Figure 2: Microscopy-based... a label control treats the sample to the full protocol without any antibodies to determine the level ...be partially addressed with stringent staining protocols and the use of isotype-specific secondary antibodies...

...permeabilization protocol, but very poorly in a methanol fixation-permeabilization protocol. Although the...Additionally, their performance depends on the specific protocol used and the abundance of the epitope in the sample...can detect the antigen of interest in the given protocol. This requires use of one of the five pillars ...labeling (or both). It is important to test for the protocol you intend to use in your experiment. An antibody...target is an important factor in deciding which protocol is likely to work, we have found that it is often...of the antibody used in the specific sample and protocol. This can be a challenge to confirm. As such, ...antibody can detect the antigen of interest in the protocol of interest, ideally at endogenously expressed...

...proteins. The Seeger lab developed a robust in-vitro protocol that enables rapid selection of synthetic nanobodies...different mRNA sybody libraries for use in the protocol, which cover 3 naturally-occurring nanobody structures...purification purposes) protein target of interest, the protocol goes through 3 major steps to select for optimal... screen for binding to the target protein. The protocol describes going through two rounds of phage display...lab used the ELISA technique described in their protocol because it is less susceptible to identifying ...sybodies (Zimmermann et al., 2020). This step of the protocol starts out with anti-Myc antibodies bound to an...the Seeger lab demonstrated they could use the protocol to rapidly identify and isolate sybodies targeting...

...3: Share protocols and spreadsheets with lab members Now that you’ve organized your protocols, wouldn’... and your lab? Organizing your lab notebooks, protocols, and to do lists can be easier than you think!..., and post-its to make finding your most used protocols & references easy to find It’s happened to all...can’t remember where you put your flow cytometry protocol. Or maybe you can’t find the molecular cloning... visibly labeled on the spine. For binders of protocols and data, consider using color coded binders and...create notes directly from webpage clippings, photographs, and file attachments. Organize your notes into...your colleagues. Start small by identifying the protocols, guides, or references that your labmates ask ...