We narrowed to 961 results for: REP
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TypeBlog Post...can vary "Lab assistants" usually wash glassware, prep media, etc. "Research assistants" are more involved...
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Tips for a 1st Time CRISPR User (by a 1st Time CRISPR User)
TypeBlog Post... to evolve over time, which is bad news for repeatability and bad news for a screen. Because Addgene viruses... -
Fluorescent Proteins 101: Photoactivatable Fluorescent Proteins
TypeBlog Post...second. The first successful irreversible PA-FP reported was PA-GFP. PA-GFP was derived from Jellyfish ... -
Cancer and the Immune System: Deciphering the Relationship
TypeBlog Post...), and ability to metastasize. There have been reports of clinical trials going amiss and patient deaths... -
Special Delivery: Fluorophore Targeting for FRET Studies
TypeBlog Post...protein kinase dynamics in living cells with FRET reporters." Methods 40.3 (2006): 279-286. PubMed PMID: 16908183... -
Chemogenetics Guide
TypeGuide...hardware-independent optogenetic inhibition. Sci Rep. 5:14366. PMID: 26399324 (Link opens in a new window...control expression of a chemogenetic reporter ensures that the reporter remains silent until a cell or tissue-specific...currently one Gs-DREADD, rM3D, that was created by replacing the corresponding intracellular region of a turkey...neurons through manipulation of ion channels arePharmacologically Selective Actuator Modules (PSAMs, pronounced...available. Browse our collection of Ready-to-use AAV Preparations of Chemogenetic Plasmids ! Table 4. Common promoters...Zhang S-C (2021). Human Stem Cell-Derived Neurons Repair Circuits and Restore Neural Function. Cell Stem... -
Optogenetics Guide
TypeGuide...light-induced inhibition of neuronal activity in vivo. Sci Rep. 5:14807. PMID 26443033 Wietek J, Prigge M. 2016....optical switches Sensors are genetically-encoded reporters of molecular signals; e.g., calcium indicators... -
CRISPR Guide
TypeGuide...sequence. The resulting DSB is then repaired by one of two general repair pathways: the efficient but error-prone...directed repair (HDR) pathway. HDR relies on copying DNA from a matching template to accurately repair or fill... NHEJ is a naturally occurring repair pathway and is the primary repair mechanism for DSBs. While this...base excision repair pathway, which fully removes the uridine from the DNA and replaces it with another... tool to activate, repress, visualize, and isolate genes. Activation or Repression of Target Genes Figure...sufficient to repress transcription by blocking initiation. More robust transcriptional repression (CRISPR ...demethylation DNMT3A - repression through cytosine methylation MQ1 - repression through cytosine methylation... -
Molecular Biology Reference
TypeGuide... Origin of Replication ( ori ) DNA sequence which directs initiation of plasmid replication (by bacteria...recombinant DNA technology, which enables the preparation of more managable (i.e., smaller) DNA fragments...double-stranded DNA that typically carry genes and can replicate independently from chromosomal DNA. Although they...the bacterial chromosomal DNA, plasmid DNA is replicated upon cell division, and each daughter cell receives...manipulating genes. For instance, most plasmids are replicated in E. coli and are relatively small (∼3000 - ...sequences for this purpose, which includes a DNA replication origin, an antibiotic-resistance gene, and a ... exists extrachromosomally in E. coli , it is replicated independently and segregated to the resulting... -
Adenovirus Guide
TypeGuide... DNA replication and host cell manipulation. Late transcripts are expressed after DNA replication starts...production of replication-competent adenoviruses (RCAs) that would be able to infect cells and replicate autonomously...Terminal Repeat, serve as self-priming structures that promote primase-independent DNA replication. 3' ITR...Terminal Repeat, serve as self-priming structures that promote primase-independent DNA replication. Homology...adenoviral vector that has the ability to replicate and reproduce within host cells as a result of a spontaneous...heavily-spliced transcripts flanked by two inverted terminal repeats (ITRs). Adenoviral genes are divided into early...were discovered as a contaminant of adenovirus preparations, which is how they got their name, but they ... -
Lentiviral Vector Guide
TypeGuide...tropism. Replication-competent The ability of a virus (or viral vector) to replicate and reproduce within...Plasmids Collection In-Stock Lentiviral Preps Addgene's Viral Prep Service Viral Vectors 101 eBook Addgene's...hosts in order to continue their lifecycle and replicate. This feature can be harnessed for stable delivery... genes, accessory genes, and the long-terminal repeats (LTRs) necessary for integration. Not all of these...of safety improvements and the potential for replication-competent lentivirus production — that is, creation...particles that could infect cells and further replicate on their own. While the viral components are still...Interchangeable; usually encodes for VSV-G Safety Safe; replication incompetent by using three separate plasmids ... -
Gamma-Retroviral Vector Guide
TypeGuide...tropism. Replication-competent The ability of a virus (or viral vector) to replicate and reproduce within...self-inactivating viral vectors. R in cis Repeat region with repeated sequence. U5 in cis Unique 5'; in some...hosts in order to continue their lifecycle and replicate. This feature can be harnessed for stable delivery...contains packaging genes and the long-terminal repeats (LTRs) necessary for integration. Wildtype gamma-retroviruses... for safety to reduce the chance of creating replication-competent retroviruses. For a summary of all ...retroviral plasmids. This will ensure that the repeats will be maintained and often results in a greater...the only type of selectable marker. Fluorescent reporters, such as GFP, are another common selection marker... -
Plan Your Experiment
TypeGuide...genome editing. Scientific Reports , 5 , 11315. https://doi.org/10.1038/srep11315 PMID: 26066060 Zukher, ...experimentally verified Cut Nick Homology Directed Repair (HDR) Generate specific genomic edits, like point...fusions with proteins that promote HDR Requires a repair template Lower editing efficiency compared to NHEJ...pegRNA optimization can be challenging Prime Edit Repress or Interfere (CRISPRi) Reduce gene expression without...sequence dCas9 or dCas9 fusion with transcriptional repressor (such as dCas9-KRAB) dCas9-KRAB can be more effective...requires donor DNA to serve as template for the repair. Once you settle on your gRNA, you can design a...target in your samples to ensure there are no discrepancies with the reference sequence you used for gRNA... -
Promoters
TypeGuide...negative or positive. Negative repressible operons, are normally bound by a repressor protein that prevents transcription...operon is normally transcribed until a repressor/corepressor binds to the operator preventing transcription...polymerase, an operator, a segment of DNA in which a repressor or activator can bind, and the structural genes...transcription. When an inducer molecule binds to the repressor, it changes its conformation, preventing its binding...of a negatively controlled operon. A positive repressible operon works in the opposite way. The operon ...bacteriophage lac Constitutive in the absense of lac repressor (lacI or lacIq). Can be induced by IPTG or lactose...Promoter of the arabinose metabolic operon trp Repressible by tryptophan Promoter from E. coli tryptophan... -
Guide to Using Pooled Libraries
TypeGuide...Introduction to Pooled Libraries Pooled libraries represent a powerful tool for forward genetic screening,... simplest level, pooled libraries are single preparations of many different plasmids. Plasmids within ...either case, next-generation sequencing of the maxiprep DNA is recommended to verify that the library ...experiments, and can also negatively affect data reproducibility. Types of Pooled Library Screens In pooled ...To make sure that every plasmid is adequately represented in the population, you’ll need to infect many...of each cell carrying it as being a biological replicate for that plasmid. Library screens can be divided...process. Amplify the library (electroporation and maxiprep). If delivering as virus, make virus; this creates... -
Cloning
TypeGuide...E. coli , where the nicks will be repaired by the normal replication process. It is important to note ...interest and then insert them into plasmids for replication and protein expression. So how do scientists ...note that restriction enzyme target sites can be repeated throughout a specific DNA sequence, which can ...creating a circular product with four nicks that are repaired by the bacteria after transformation. LIC does... has difficulty assembling DNA fragments with repetitive sequences and DNA that ends in sequences that... -
Antibody Guide
TypeGuide... engineered small proteins derived from ankyrin repeat proteins. These are antibody mimics instead of ...isotype. Overview of application Fix your samples. Prepare your samples using permeabilization, antigen retrieval...and controls follow that of IF (above). Sample preparation, signaling excitation, and assay readout vary...must also be run. Unique samples (non-biological replicates) often each require their own set of controls...conjugating them in the lab? How will the sample be prepped, and will this or other factors affect epitope ... -
Sequencing Primers
TypeGuide...distribute primers. For sequencing plasmids in our repository, we've chosen primers based on the plasmid backbone...Qiagen) 5' of MCS in pQE vectors, forward primer pREP Forward GCTCGATACAATAAACGCC (Invitrogen) Rous sarcoma... -
Educational Resources
TypeGuide... How-to Blog Read about plasmids in Addgene’s repository , and related molecular biology techniques. Topics... -
Science Guides
TypeGuide...Pooled Libraries Pooled libraries are single preparations of many different plasmids. Plasmids within ...