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...this stage, only cells from the immune system show wide scale mobility with movement from the bone marrow...you can detect signal in thick specimens. See the video below for an example of two-photon intravital microscopy... in diverse tissue environments. These studies provide quantitative measurements of cellular motility,...and skin. Two-photon intravital microscopy. The video above depicts the infiltration of neutrophils expressing...technical specificities have to be taken into consideration especially in the use of fluorescent proteins... Addgene.org Check out Our Fluorescent Protein Guide Pages Find Plasmids for Subcellular Localization...

...scarless DNA constructs using homologous regions to guide the joining reaction. BioBrick Assembly will leave...fragments, and generate constructs for producing single-guide RNAs – but it also allows you to skip purification...NEBuilder HiFi so accurate (apart from the high-fidelity polymerase) is the ability to remove, reset and...freely available online. Its simple interface can guide you through both experimental and primer design ...single-stranded synthetic oligo to generate a single-guide RNA (sgRNA). Using only one single-stranded oligo...

...Nicholas T., et al. “Genome-wide analysis in vivo of translation with nucleotide resolution using ribosome...sequenced and aligned to the genome to determine genome-wide translational frequency. Although highly useful,...reporter mRNAs. Using translation inhibitors cycloheximide and puromycin, Halstead et al. verified that... translated, the SunTag sites on the nascent polypeptide recruit scFV-GFP, generating green puncta that...

...smaller fragments that are then barcoded for identification. This mixture of barcoded fragments is known...for each sample remain highly normalized across a wide range of input DNA concentrations. This process ...of 96 plasmids on a plate) will typically have a wide range of input quantities. This variation is problematic...are produced from large numbers of samples with a wide range of input concentrations (via proprietary chemistry...alternative to Sanger sequencing because the sample identity information is always displayed at the individual...

...situation. But a few days later we had an idea. And this idea rapidly grew and became Crowdfight COVID-...modern science in our view. And it was this very idea that research is done better with others than by...has been essential to push certain projects. The idea behind the platform was simple: any person working...remote collaborations Read our Transferable Skills Guide article about external collaborations Resources...

...physical location didn’t matter. I didn’t have to consider that aspect and I could plan more with regard ...depending on the conference, the audience can help decide which questions get answered. “You can upvote a...presenting is eye contact. “It’s important to keep your video on and look into the camera to make it seem more...on Twitter where each poster presenter had four “slides” to use for their poster, eliminating the need ...to make space for it,” says Bachle. “On the flip side, you can attend conferences for just a little bit...

... the widely used 293T. During vector production, assembled AAV vector particles accumulate inside the ...either associated with the outside surface of the exosome or are carried inside the vesicle, which may also...Addgene's viral service Find viral protocols and videos Find AAVs for your research ...

...working in academia or industry. At BosLab, we identify ourselves as part of a network of independent ...square, but we saw the potential in the space and decided to reactivate it. We started out by holding a few...from hacking probiotics to filming educational lab videos, while some are looking to develop new skill sets... mind. Thus, to encourage more involvement and provide all our members the opportunity to do molecular...project. This project was inspired by previous city-wide genomic studies and our study will involve getting...

...product because the plasmids in the library are identical except for the insert; there’s no need to sequence...results represent a successful amplification? To provide a point of reference, Addgene commonly prepares...results If your sequencing is successful, your NGS provider should return results in the form of many, many... want! At the end of the day, it’s up to you to decide your threshold of what a good amplification looks...post has helped clarify some of the important considerations when using, amplifying, and verifying pooled...

...efficiencies and expression levels in the cells. Ideally, the chosen reporter is unique to the cell, is ... generations. Stably transfected cells may be considered an entirely new cell line from that of the original..., NIH3T3, CHO, 293HEK, Jurkat T cells Blocks polypeptide synthesis at 80S; inhibits chain elongation 100...hygB HeLa, NIH3T3, CHO, Jurkat T cells Blocks polypeptide synthesis at 80S; inhibits chain elongation. ... 10-14 days depending on how quickly your cells divide. Gentamicin is often used as a supplement in mammalian...

...fall back to? We have outlined some important considerations to help you choose the plasmid prep kit that...or by incorporating recycled paper and plastic. Aside from the more obvious swaps like using smaller bottles...Where can you look for help?  All manufacturers provide specs, protocols, and quality control information...help explain the rationale behind each step and provide troubleshooting advice. In these cases, it can ...between these manufacturers, as price is a large consideration for most labs. Many institutions have preferred...department for these.  While budget is an important consideration, you do want to make sure you’re getting the...

...chemical substrate (and sometimes cofactors) to be provided and are generally dimmer than fluorescent proteins... remains popular, other luciferases have been identified in the sea pansy (Renilla reniformis), click ...assays (low background, no photobleaching) but provides flexibility by pairing the luciferase with a variety...change in conformation of a single protein. Considerations for luciferase assays Luciferases can only ...luciferase output at different wavelengths. Addgene provides empty backbones with the luciferase gene, a variety...

...of dNTP in the reaction mix, the researcher can decide exactly how far the T4 exonuclease proceeds: at...at the first occurrence of the chosen nucleotide, T4 will switch to polymerizing and then stall due to...the “damage” and producing a complete plasmid. Besides being compatible with any vector, the SLIC protocol...multi-fragment DNA assembly, and its low cost makes it ideal for researchers doing large amounts of cloning. ... 10.1007/978-1-61779-564-0_5. PubMed. JBEI-J5’s guide to SLIC Resources at Addgene Ligation-Independent...

...particular marker, you can use the reference plots provided by the antibody companies or check the literature...sample. The isotype antibody will be virtually identical to the antibody for your marker of interest. It...for that particular marker. This enables you to identify any non-specific binding to know that your positive...unsure what amount of antibody to use, you should consider using an isotype control. Pro tip! Many companies...Cytometry Resources on Addgene.org Addgene's Antibody Guide Browse Addgene's Antibody Collection        ...

...Visit Our AAV Guide Page Find Ready-to-use Virus for Your Research Visit Our Lentivirus Guide Page ...understand brain function So far scientists have identified 180 brain regions. Both this geography and the...Using retrograde AAV AAVrg can be used to deliver a wide array of genetic tools for neural interrogation ...where the cell bodies of the projection neurons reside.   Impact for scientists While the mechanisms ...

...protein. Animals are first injected with a small peptide that represents a short, unique amino acid sequence...isolate individual hybridomas and ELISA is used to identify the hybridomas producing the most specific antibodies...antibody, meaning that the antibodies are largely identical between lots.  Disadvantages of monoclonal antibodies...antibody-encoding genes over time as the hybridoma cells divide. These changes can alter the antibody that is actually...produced by the hybridoma. Any genetic drive can be identified by sequencing the heavy and light chains. To ...

...suggests a change to a slide, think about whether the same principle applies to other slides in your deck.  If... craft sentences for abstracts and presentation slides, learn from audiences as they do science outreach...of a new science communication initiative that provides yet more scientists with opportunities to practice...Resources on Addgene.org Check out how Addgene uses videos for science communication ...

...in the lab. This is commonly done using calcium chloride which permeabilizes the cell membrane so the bacteria...inject their genetic material into the cell. Once inside, phages can follow one of two different life cycles...both of these life cycles bacterial DNA can be accidentally packaged into the newly created phages. Transfer...referred to as transduction. Transferred DNA once inside the infected bacterium can either exist as transient...Addgene’s protocol page, which has both protocols and videos for techniques in basic molecular biology, plasmid...

...more OptoNBs would allow for more uses both inside and outside cells. References Gil, Agnieszka A., et...exciting because using light to control signaling provides greater spatiotemporal resolution than pharmacological...domain of the Sons of Sevenless (SOS), a guanine nucleotide exchange factor that activates Ras. They expressed...more about optogenetics in Addgene's optogenetics guide ...

.... To make a point mutation, you design two oligonucleotide primers with the desired mutation, then use...3-4 phage proteins are needed and these can be provided via a separate plasmid or be inserted into the...insertions and deletions. The figure above shows guidelines for primer design; a desired insertion should...REPLACR-mutagenesis successfully incorporated a 60-nucleotide insertion; beyond this insertion size, primer...introduce mutations. Using commercially available high-fidelity polymerases such as KOD and Phusion, which are...