...discussion. The goal of the presentation is to: Give a general overview of the technology and how it can be used...

...PMID: 31084567 . The content for this page was generated with the help of Richard Kahn . Browse ARF GTPase...

...mammalian systems, Cas13a does not exhibit the collateral RNA degradation seen in bacteria. ID Plasmid ...

...SYFP2 (yellow fluorescent protein) in striatal and lateral septum cholinergic neurons Jonathan Ting Viviana...

... Promoter Measure promoter strength pBV-Luc - Luciferase reporter plasmid with very low...

...endogenous zebrafish genes; many of these were generated as part of an NIH-sponsored effort to provide ...

... you have a genome with unknown sequence. In the era before sequencing was commonplace, researchers used... ultimately leading to what are generally considered first generation DNA sequencing machines (9). Today... amount of high-quality DNA, real-time PCR has several advantages including easier automation, higher ...both time and resources (3). Still, researchers generating transgenic animals may choose Southern blotting.... Today, researchers use both Sanger and next-generation sequencing techniques to answer similar experimental... subset to sequence. Sequencing gels and next-generation sequencing techniques Introduced in 1977, Sanger...samples at once for later comparison. These next-generation sequencing techniques do not typically rely on...

.... Why an International Mentorship Program? In an era where millions of people hold a PhD, achieving our...people and society. Future generations of IMP Motivating future generations is key to the success of this...world, professional internationalization faces several obstacles; for instance there are few resources...Programs at BWH and providing career mentoring to several students from Spain and all over the world, she...the country. Brain drain is a serious problem in several countries, however programs like IMP, bring closer...from international financial struggle, our new generations feel thrown, to sink or swim on their own, into...Development, and Innovation, D. Carmen Vela, who reiterated the Spanish government’s support for the IMP ...

...Retrograde AAV *The AAV retro helper plasmid was generated as a part of the Janelia Research Campus viral...

...choosing your AAV capsid. Your choice could depend on several factors including: the delivery method, mouse strain...

...window) was established to advance research and therapies for brain disorders by enabling region- and cell-specific...

...BGHpA AiP20013 AiE2674m SYFP2 Entorhinal area, lateral part - L2 stellate neurons Hippocampal formation...

...threshold of a revolution in modern neuroscience, an era in which technologies like FLEx and optogenetics ...arousal in mice previously subjected to steady-state general anesthesia (Taylor et al., 2016). These very same...modulation of FLEx vectors combined with the use of several Cre transgenic mice targeting different brain areas...PMCID: PMC6317997. Deisseroth, Karl, et al. "Next-generation optical technologies for illuminating genetically...exceeding the carrying capacity of AAV vectors." Gene Therapy for Neurological Disorders. Humana Press, New York...26135556. Hudry, Eloise, and Luk H. Vandenberghe. "Therapeutic AAV gene transfer to the nervous system: a clinical...ventral tegmental area induces reanimation from general anesthesia." Proceedings of the National Academy...

...technologies need to be discarded but clearly we are in an era of neuroscience research where optogenetics will ...methodological and experimental limitations and considerations? I recently spent two months in the lab of ... at NIDA and some of the practicalities and considerations you may not find in the materials and methods...prove that your optogenetic manipulations are generating action potentials (or lack there of, depending...hyperpolarization, and inhibition of action potential generation. Archaerhodopsin (Arch) is activated at a similar... no longer available through Kopf). Additional general protocols on stereotaxic surgeries for viral injections...are obviously limited by the mouse line itself. Generation, breeding, and maintenance of a mouse line is...

... master mix preparation and droplet generation (droplet generation BSC) to ensure no contamination of ...cap the PCR tubes. Generate the Droplets Bring the PCR tubes to the droplet generation BSC. Without disturbing...holder to the droplet generator. Close the lid and wait for the droplets to be generated. Once the droplets...is not at temperature, touch the screen on the plate sealer to allow it to get to temperature. Once the...increase the accuracy of the titer, take the average of several measurements. Figure 1: ddPCR sample data...12–5E+13 GC/mL, where GC is genome copies and generally represents the physical number of viral particles...surfaces with 10% bleach. Safety Warnings AAV is generally considered biosafety level 1 but may require BSL...

...for generating stable cell lines with lentivirus . Day 0: (optional) Seed cells for the generation of ...theory a monoclonal line could be generated at any time. Considerations Before You Start Some cell lines...number of monoclonal lines generated using this protocol will vary based on several factors, including the...seeding the plate at an average density of 0.5 cells/well. Seeding an average of 0.5 cells/well ensures...This protocol describes how to generate a monoclonal cell line from a polyclonal pool of stable cells...Service Introduction This protocol describes how to generate a monoclonal cell line from a polyclonal pool ...expressing clones take over the polyclonal cell pool. Generating a monoclonal cell line by limiting dilution results...

...science. General Considerations The ELISA can be completed in a single day or broken up over several days ...of our protocols supports reproducibility and accelerates science. Here, we list the specific equipment...incubating certain steps at 4 °C instead of room temperature or 37 °C. The protocol notes when there are different...1L Foil Before Starting Warm reagents to room temperature. Procedure Section 1: Prepare the Antigen Standard...into 999.5 mL 1X PBS Cap the bottle and invert several times to mix. Carefully remove the plate seal from...of BSA to 25 mL of PBS. Cap the tube and invert several times to mix. Using a multichannel pipette, add...microplate shaker set to 400 rpm for 2 h at room temperature or overnight at 4 °C . Section 3: Primary antibody...

...holder to the droplet generator. Close the lid and wait for the droplets to be generated. Once the droplets...is not at temperature, touch the screen on the plate sealer to allow it to get to temperature. Once the...increase the accuracy of the titer, calculate an average of several dilutions. Figure 1: ddPCR Lentivirus sample...1E+09 TU/mL, where TU is transducing units and generally represents the number of infectious viral particles...with 10% bleach. Safety Warnings Lentivirus is generally considered biosafety level 2+. Please ensure that...Supermix for Probes no dUTP, Bio-Rad,1863023 Droplet generation oil, Bio-Rad, 1863005 DG8 cartridge, Bio-Rad,...tubes. Pipette back and forth 10 times to mix. Generating the Droplets Place a DG8 cartridge into the cartridge...

...ligation. Because of its dual polymerase/exonuclease functions, T4 DNA polymerase can create overhangs of varying... Because the polymerase reaction is favored over the exonuclease reaction, the polymerase will add back...room temperature, then add 1 μl of 25 mM EDTA, followed by another 5 minutes at room temperature. The ... of the enzyme and shift its activity back to polymerase. In our case, dGTP will be used in the reaction...primers will have different leader sequences, but operate on the same principle (homologous to the first ...web-based primer design software to ensure a melting temperature between 50-60°C for your PCR primers. Step 2:...Overhangs Treat the linearized vector with T4 DNA polymerase to "chew back" the free 3' ends, following the...

...Human U6 promoter drives RNA Polymerase III transcription for generation of shRNA transcripts. cPPT Central...minutes then slowly cool to room temperature over the period of several hours. If using a beaker of water... any kind regarding the contents of any literature. Literature are provided to you as a guide and on an...complex in the transduced cells. hPGK Human phosphoglycerate kinase promoter drives expression of puromycin...information, visit Addgene’s Mammalian RNAi Tools page. Several other laboratories have deposited pLKO derived ...because polyT is a termination signal for RNA polymerase III. Note that these were the prevailing guidelines... B.2 Ordering Oligos Compatible with pLKO.1 To generate oligos for cloning into pLKO.1, insert your sense...